Compounds and methods of use to treat infectious diseases

ABSTRACT

The present invention concerns alkyl aryl carbonyl compounds that possess anti-infective activity. The compounds of the invention can be used to target specific nuclear localization signal, thereby blocking importation of specific proteins or molecular complex into the nucleus of a cell. The invention encompasses methods of use of such compounds for treatment or prevention of infectious diseases, such as parasitic and viral diseases, including, for example, malaria and acquired immunodeficiency syndrome. The use of the compounds to detect certain specific protein structures which are present in nuclear localization sequences is also taught.

1 FIELD OF THE INVENTION

[0001] The field of the present invention concerns compounds that reactwith specific sequences in proteins. The present invention moreparticularly concerns a class of compounds that react, under physiologicconditions, with proteins having adjacent or neighboring basic aminoacid sequences. The compounds of the invention can be used to labelspecifically such proteins for research purposes and to disrupt theirfunction for pharmacologic purposes. The compounds of the invention canbe used for targeted inactivation of nuclear localization signal inspecific proteins or molecular complexes. The compounds of the inventioncan also be used to treat infectious diseases such as HIV infection andmalaria.

2 BACKGROUND TO THE INVENTION 2.1 THE DERIVATIZATION OF PROTEINS

[0002] Those skilled in the art will appreciate that there are manycompounds that can react with specific amino acid residues in proteins,e.g., with sulfhydryl, amino, carboxyl moieties. These reagents aresubstrate specific, in the sense that each reacts only with one or a fewspecific amino acids wherever they occur within a protein's sequence.

[0003] However, the reactivity of such reagents is not affected by theadjacent or neighboring amino acids that form the environment of thereactive moiety. Thus, the reactivity of such compounds is not contextor neighborhood specific.

2.2 NUCLEAR IMPORTATION

[0004] The function of an intracellular protein is usually the result ofthe overall three dimensional (tertiary) structure of the protein.However, nuclear importation is determined by the simple presence of ashort sequence, called a nuclear localization signal (NLS), whichfunctions relatively independently of its position relative to theremainder of the structure of object that is imported. In eukaryoticcells all proteins are made in the cytoplasm, which is outside of thenucleus. In general, those proteins larger than 40 kD that arespecifically localized in the nucleus of the cell must be activelyimported into the nucleus through the nuclear membrane from thecytoplasm via an ATP-dependent mechanism that is independent of celldivision. The proteins, and other objects, that are imported have anuclear localization signal (NLS), usually located within the NH₂terminal segment of the protein. Several such sequences are known:

[0005] a. PKKKRKV from large T antigen of SV40 and otherpapillomaviruses such as JC, see Kalderon, D., et al., 1984, Cell39:499-509;

[0006] b. [AV]KRPAATKKAGQAKKKK [LD] from nucleoplasmin, in which onlyone of the two bracketed sequences is required, Dingwall, C., et al.,1988, J. Cell Biol. 107:841-49;

[0007] c. PRRRRSQS from hepatitis B HbcAg-Yeh, C. T., 1990, J. Virol.

[0008] d. KRSAEGGNPPKPLKKLR from the retinoblastoma gene productp110^(rb1)-Zacksenhaus E. et al., 1993, Mol. Cell. Biol. 13:4588

[0009] e. KIRLPRGGKKKYKLK from the matrix protein of HIV-1, Bukrinsky,M. I., et al., 1993, Nature 365:666.

[0010] Other viruses that contain NLS sequences include influenza virus(NP, PA, PB1, PB2 proteins which have lysine-rich NLS similar to SV40),hepatitis delta virus (HDAg, which has the sequence PKKKXKK),parvoviruses such as RA1 (NS, VP proteins which have lysine-rich NLSsimilar to SV40), Herpes simplex and measles virus. The recognition ofan NLS sequence is largely independent of the detailed structure of theobject which includes it and of its site of attachment. Goldfarb, D. S.et al., 1986, Nature 332:641-44; Lanford, R. E., 1986, Cell 46:575. Merejuxtaposition of the amino acids of the NLS is not sufficient forfunction, for example NLS function is generally not conferred by thepeptide having the same sequence of amino acids in the opposite order asthe NLS sequence. Adam, S. A. et al., 1989, Nature 337:276-79.

[0011] The primary structure, i.e., the linear sequence, of the NLS mostfrequently contains consecutive lysines, the N^(ε) moieties of whichpresumably closely approach one another, i.e., they are neighbors.However, certain functional NLS peptides lack consecutive lysines.Robbins, J., et al., 1991, Cell 64:615-23. Presumably the secondary andtertiary structure of these so called “bipartite” NLS peptides givesrise to neighboring N^(ε) moieties, which may be important for theiractivity.

[0012] Docking and subsequent movement of proteins across the nuclearpore complex require transport factors. Import of NLS-containingproteins across the nuclear pore complex is mediated by karyopherin αβheterodimers (also termed NLS receptor/importin) which bindNLS-containing proteins in the cytosol and target them to the nucleus(Gorlich, D., et al., 1995, Curr. Biol. 5:383-392; Radu, A., et al.,1995, Proc. Natl. Acad. Sci. 92:1765-1773). Karyopherin a binds the NLS(Adam and Gerace, 1991, Cell 66:837-847) whereas karyopherin β enhancesthe affinity of α for the NLS (Rexach and Blobel, 1995, Cell 83:683-692)and mediates docking of karyopherin-NLS protein complexes tonucleoporins (a collective term for nuclear pore complex proteins) thatcontain FXFG peptide repeats. The GTPase Ran and its interacting proteinp10 (also termed NTF2) (Moore and Blobel, 1994, Proc. Natl. Acad. Sci.91:10212-10216) impart mobility to the translocation process bycatalyzing the disruption of karyopherin αβ heterodimers that havedocked to a nucleoporin (Nerhbass and Blobel, 1996, Science272:120-122). Partial reactions of the nuclear import can be reproducedin vitro using solution binding assays and recombinant karyopherins(Rexach and Blobel, 1995, supra).

[0013] Two inhibitors of the nuclear localization process have beendescribed. Nuclear localization has been inhibited by lectins (e.g.,wheat germ agglutinin (WGA)) that bind to the O-linked glycoproteinsassociated with nuclear localization. Dabauvalle, M. -C., 1988, Exp.Cell Res. 174:291-96; Sterne-Marr R., et al., 1992, J. Cell Biol.116:271. The nuclear localization process, which also depends upon thehydrolysis of GTP, is blocked by a non-hydrolyzable analog of GTP, e.g.,(γ-S)GTP, Melchior, F., 1993, J. Cell Biol. 123:1649.

[0014] However, neither (γ-S)GTP nor WGA can be used as pharmaceuticals.Proteins, such as WGA, can be introduced into the interior of a cellonly with considerable difficulty. The same limitation applies tothiotriphospates such as [γ-S]GTP. Further, GTPases are involved in amultitude of cell processes and intercellular signaling, thus, the useof a general inhibitor of GTPases would likely lead to unacceptable sideeffects.

2.3 THE SIGNIFICANCE OF NUCLEAR IMPORTATION IN HIV-1 INFECTIONS

[0015] Although HIV-1 is a retrovirus, it and other lentiviruses must bedistinguished from viruses of the onco-retrovirus group, which are notassociated with progressive fatal infection. For example, lentivirusesreplicate in non-proliferating cells, e.g., terminally differentiatedmacrophages, Weinberg, J. B., 1991, J. Exp. Med. 172:1477-82, whileonco-retroviruses, do not. Humphries, E. H., & Temin, H. M., 1974, J.Virol. 14:531-46. Secondly, lentiviruses are able to maintain themselvesin a non-integrated, extrachromosomal form in resting T-cells.Stevenson, M., et al., 1990, EMBO J. 9:1551-60; Bukrinsky, M. I., etal., 1991, Science 254:423; Zack, J. L., et al., 1992, J. Virol.66:1717-25. However, it is unclear whether this phenomenon is related tothe presence of latently infected peripheral blood lymphocytes (PBL) inHIV-1 infected subjects, wherein the virus is present in a provirusform. Schnittman, S. M., 1989, Science 245:305; Brinchmann, J. E., etal., 1991, J. Virol. 65:2019; Chapel, A., et al., 1992 J. Virol.66:3966.

[0016] The productive infection of a cell by a retroviruses involves thesteps of penetration into the cell, synthesis of a DNA genome from theRNA genetic material in the virion and insertion of the DNA genome intoa chromosome of the host, thereby forming a provirus. Both lenti- andoncoretroviruses gain access to the host cell's nucleus during mitosiswhen the nuclear membrane dissolves. However, the lentiviruses are alsoable to cross the nuclear membrane because viral proteins containingnuclear localization sequences are associated with the viralnucleoprotein complex.

[0017] The productive infection of terminally differentiated macrophageslocated in the central nervous system is thought to be responsible forthe dementia associated with AIDS. Keonig, S., et al., 1986, Science233:1089; Wiley, C. A. et al., 1986, Proc. Natl. Acad. Sci. 83:7089-93;Price, R. W., et al., 1988, Science 239:586-92. The infection ofterminally differentiated macrophages in the lymphoid system is known tocause aberrant cytokine production. Guilian, D., et al., 1990, Science250:1593; Fauci, A. S., et al., 1991, Ann. Int. Med. 114:678. Thus, thewasting syndrome associated with HIV-1, also known as “slim” disease, isbelieved to be a pathological process that is independent of the loss ofCD4-T-cells. Rather the pathobiology of the wasting is closely relatedto the pathobiology of cachexia in chronic inflammatory and malignantdiseases. Weiss, R. A., 1993, Science 260:1273. For these reasons, theinhibition on HIV-1 infection of macrophages and other non-dividingcells is understood to represent a highly desired modality in thetreatment of HIV-1 infection, especially for patients wherein dementiaor cachexia dominate the clinical picture.

[0018] Macrophages play an important role in the transmission of HIV aswell. During early stages of the infection, macrophages and cells of themacrophage lineage (i.e. dendritic cells) may be the primary reservoirof HIV-1 in the body, supporting infection of T cells by antigenpresentation activities, Pantaleo, G., et al., 1993, Nature 362:355-358,as well as via the release of free virus. Direct cell-to-celltransmission of the virus may constitute the major route by whichinfection spreads during the early-stages of the disease, afterresolution of the initial viremia.

[0019] It is noteworthy, in this regard, that macrophage-tropic strainsof HIV-1 predominate in the early stages of infection. Thus, it appearsthat the infection of macrophages is particularly important during thedevelopment of a chronic infective state of the host in a newly infectedsubject. Secondly, macrophages are the HIV-susceptible cell type mostreadily passed during sexual intercourse from an HIV-infected individualinto the circulation of an uninfected individual.

[0020] Finally, infection of quiescent T cells by HIV-1 has been shownto take place in vitro, Stevenson, M., et al., 1990, EMBO J.9:1551-1560; Zack, J. A., 1990, Cell 61:213-222, and probablyconstitutes an important pathway for the spread of infection in vivo atvarious stages of the disease. Bukrinsky, M. I., et al., 1991, Science254:423-427. Although HIV-1 does not establish productive replication inquiescent T cells, the extrachromosomal retroviral DNA can persist inthe cytoplasm of such cells for a considerable period of time, andinitiate replication upon activation of the host cell. Stevenson, M., etal., 1990, EMBO J. 9:1551-1560; Spina, C. A., et al., 1994, J. Exp. Med.179:115-123; Miller, M. D., et al., 1994, J. Exp. Med. 179:101-113. Arecent report suggests that the duration of viral persistence in thequiescent T cell depends on the presence of a functional NLS. vonSchwedler, U., et al., 1994, Proc. Natl. Acad. Sci. 91:6992-6996. Thus,physicians recognize the desirability of preventing the infection ofmacrophages by HIV and understand that substantial benefits would beobtained from the use of a pharmacologic agent that prevents HIVinfection in this cell type.

[0021] The mechanism whereby HIV, but not oncoretroviruses, infectnon-dividing cells is now understood in broad outline. It is establishedthat the function of the pre-integration complex of retrovirus in thisregard does not depend upon the cellular mechanisms of mitosis or DNAreplication, per se. Rather the integration complex must merely gainaccess to nucleus. Brown, P. O., et al., 1987, Cell 49:347.Onco-retroviruses gain access to the nucleus upon the dissolution of thenuclear membrane in mitosis. By contrast, lentiviruses contain twodistinct proteins that mediate nuclear access through the nuclear porecomplex in the absence of cellular division. For the first of these, thematrix protein (MA or p17), nuclear importation activity is clearly dueto the presence of a trilysyl-containing NLS sequence. Bukrinsky, M. I.,et al., 1993, Nature 365:666; von Schwedler, U., et al., 1994, Proc.Natl. Acad. Sci. 91:6992. A second protein subserving the function ofnuclear entry, the vpr protein, does not contain an identifiable NLSconsensus sequence. Emerman, M., et al., 1994, Nature 369:108;Heinzinger, N. K. et al., 1994, Proc. Natl. Acad. Sci. 91:7311.

[0022] The significance of the NLS sequence in the importation of HIV-1into the nucleus of non-dividing cells has been illustrated inexperiments wherein the presence in the medium of a high concentration(0.1 M) of the peptide having the sequence of the SV40 T-antigen NLSblocked the importation of HIV-1 into the nucleus ofaphidicolin-arrested CD4⁺ MT4 cells. Gulizia, J., et al., 1994, J.Virol. 68:2021-25.

2.4 INFECTIOUS DISEASES AND ITS TREATMENT

[0023] Treatment of an infectious disease with chemicals involveskilling or inhibition of growth of the infectious agent, which mayinclude free-living and parasitic organisms. Parasitic diseases arewidespread in the animal world where a parasitic organism lives at theexpense of a host organism, and causes damage, or kills its host.Humans, domestic pets and livestocks are hosts to a variety ofparasites. Parasites do not comprise a single taxonomic group, but arefound within the protozoans and metazoans, among other groups. In manyways, infectious parasitic diseases resemble infectious diseases causedby microbiologicals such as fungi, bacteria and viruses.

[0024] Malaria remains one of the major health problems in the tropics.It is estimated that 300 million people a year are infected with malaria(World Health Organization, 1990, Malaria pp.15-27. In TropicalDiseases, Progress in Research 1989-1990, Geneva). Malaria istransmitted by Anopheles mosquitos in endemic areas, and often by bloodtransfusion in eradicated areas.

[0025] Malaria in humans is caused by at least four protozoan species ofPlasmodium: P. falciparum, P. vivax, P. ovale and P. malariae. Theasexual erythrocytic parasite, merozoite, is the stage in the life cyclethat causes the pathology of malaria with a characteristic pattern offever, chills and sweats. Anemia, acute renal failure and disturbancesin consciousness are often associated with malarial infection. P.falciparum can produce a large number of parasites in blood rapidly, andcauses the most morbidity and mortality.

[0026] The most important treatment of malaria to date is chemotherapyusing a number of natural and synthetic drugs. Antifolates, such aspyrimethamine, inhibit the parasite's dihydrofolate reductase, whereasthe aminoquinolines, such as chloroquine (4-aminoquinoline) have thedigestive vacuoles as their major site of action. Prior to theintroduction of chloroquine in the 1940's, quinine was the onlyeffective drug for treatment of malaria. Chloroquine is commonly used totreat acute infections with all four species, but has no effect onrelapses of infection by P. vivax or P. ovale.

[0027] Chloroquine (500 mg weekly) may also be used to prevent malariaby suppressing the stages that multiply in the erythrocytes and causethe symptoms.

[0028] However, the use of these drugs in certain areas and in thefuture will be seriously hampered by the emergence of drug resistantparasites. Chloroquine resistance is widespread and will continue toappear in new areas. Due to the possibility of resistance, the presenceof parasites in blood (i.e., parasitemia) is followed closely duringtreatment, and alternative drugs instituted if indicated. The decisionon drug regimen will depend on the origin of the infection. Combinationtherapy, such as quinine and Fansidar (pyrimethamine and sulfadoxine),is applied to treat chloroquine-resistant P. falciparum. Because of thepresence of multidrug resistant P. falciparum in many parts of theworld, prevention of malaria by chemoprophylaxis with currentlyavailable drugs is not always effective.

[0029] In the last 20 years, only several drugs, such as mefloquine,halofantrine and artemisinin derivatives, have been developed to treatP. falciparum (Nosten et al., 1995, Drug Saf. 12:264-73). In view of thecontinuing spread of multidrug resistant P. falciparum, it is apparentthat novel effective chemotherapeutic agents are needed for use againstmalaria.

3 SUMMARY OF THE INVENTION

[0030] The present invention encompasses a class of alkyl aryl carbonylcompounds that forms stable binding interactions, preferably throughformation of reversible covalent bonds, with one or more basic aminoacid residues, wherein such basic amino acid residues are a part of anuclear localization signal (NLS). The stable binding interactionresults in the inhibition or neutralization of the nuclear localizationactivity of the NLS. The binding interaction is mediated by onefunctional component of the compound, i.e., the reactive group, whereasanother functional component of the compound, i.e. the targetting group,determines the specificity of the compound for different-NLS.

[0031] This targetting function occurs by interaction of the targettinggroup with a docking site that is positioned proximately to thesusceptible basic residues of the target NLS, such that docking of thecompound places it in a favorable configuration to form a stableinteraction with basic amino acid residues of the target NLS. Thedocking site is located either on the same NLS-bearing protein, or onanother component of a larger molecular complex that includes theNLS-bearing protein.

[0032] Preferred compounds of the invention provide divalent arylcarbonyl moieties as the reactive group, particularly aryl bis(ketone),aryl bis(α-diketone) or aryl bis(β-diketone), linked to a targettinggroup, preferably to a nitrogen-containing heterocyclic functionalityvia an N-linkage. Particularly preferred compounds provided are bisacetyl, propanoyl, glyoxyloyl, pyruvoyl, 2-oxobutanoyl, acetoacetyl,3-oxopentanoyl, 3-oxo-2,2-dimethylbutanoyl or3-oxo-2,2-dimethylpentanoyl substituted aniline moieties N-linked to apyrimidinium, pyrimidine or triazine moiety.

[0033] The invention further encompasses methods of using the compoundsof the invention to form tandem binding interactions with proteinshaving neighboring basic residues. As used, herein, neighboring basicresidues are two basic amino acid residues of a protein, particularlylysine and arginine residues, the side chain amino or guanidinofunctions of which approach each other as closely as the bis carbonylfunctions of the arylene bis(carbonyl) compounds of the invention, whenthe protein is in its natured conformation. As used herein neighboring,adjacent and juxtaposed are equivalent terms in reference to amino orguanidino moieties, and refer to the physical locations of the amino orguanidino moieties in the structure of the native protein and not to thepositions of the basic amino acid residues themselves in the linearsequence.

[0034] In one embodiment of the invention, the compounds of theinvention can be used to react with neighboring nitrogenous moieties inlysine and arginine residues of nuclear localization sequence (NLS) of aprotein, thereby inactivating the NLS. The invention also encompassesuses of compounds of the invention for specifically inhibitingimportation of a NLS-containing protein or molecular complex comprisinga NLS-containing protein, into the nucleus of a cell. The carbonylgroup(s) of the compounds of the invention inactivates the NLS of aprotein which is essential for nuclear translocation of the complex. Thecompounds of the invention are targeted to the complex by the specificinteraction between the targetting group, preferrably anitrogen-containing heterocyclic targetting group, and a docking site ona molecule in the complex that is distinct from the NLS. The inventionfurther encompasses methods of screening for alkyl aryl bis(carbonyl)and bis(dicarbonyl) compounds that are capable of inhibiting importationof a specific NLS-containing protein or molecular complex comprising aNLS-containing protein into the nucleus of a cell. The screening assaysof the present invention allow a compound of the present invention to beidentified and selected without advance knowledge of the specificdocking site that confers specificity on the NLS inhibitory activity ofthe compound.

[0035] The invention further encompasses methods of inhibitingproductive infection by HIV-1 of terminally differentiated (non-dividingcells), particularly macrophages, by inhibition of the importation ofthe cytoplasmic HIV-1 complex into the nucleus of cell. Particularly theinvention concerns the administration of the compound effective to blocksuch importation to a cell. Thus, in one embodiment, the inventionencompasses methods of using the above-described compounds to preventproductive infection of terminally differentiated macrophages andresting T-cells in HIV-1 infected subjects.

[0036] Without limitation as to theory, the compounds of the inventionis believed to block HIV-1 replication by binding to reversetranscriptase and formation of tandem Schiff bases with neighboringN^(ε) moieties of lysines in the nuclear localization signal of HIVmatrix antigen. As a result, the matrix antigen is unable to interactwith karyopherin α of the host cell and the viral nucleoprotein complexdoes not pass across the nuclear membrane via interaction with thenuclear pore transport complex and/or other cellular components.

[0037] Moreover, compounds of the present invention are also useful forinhibiting viral infection or nuclear translocation of viral proteins inproliferating cell populations, to the extent that such occurs in someof the cells in the population during periods of the cell cycle in whichthe nuclear membrane is intact. Such infection or nuclear translocationof proteins in a proliferating population of cells is susceptible totreatment with the compounds of the present invention on the same basisas non-dividing or quiescent populations would be susceptible.

[0038] The invention further encompasses methods of using the compoundsof the invention in treating or preventing infectious diseases such asthose caused by parasites, particularly Plasmodium species that causemalaria.

4 BRIEF DESCRIPTION OF THE FIGURES

[0039] FIGS. 1A-C. The structures of exemplary Compounds No. 2, 11 and13 are, respectively, FIGS. 1A, 1B, 1C.

[0040] FIGS. 2A-C. The effect of various concentrations of Compound No.2 on RT activity in the supernatant of HIV-1-infected monocytes. FIG.2A: Multiplicity of Infection (MOI) 1 ng p24/10⁶ monocytes, cultured inpresence of M-CSF. FIG. 2B: MOI 8 ng p24/10⁶ monocytes, cultured inabsence of M-CSF. FIG. 2C: MOI 0.8 ng p24/10⁶ monocytes, cultured inabsence of M-CSF.

[0041]FIG. 3. The effect of various concentrations of Compound No. 2 onRT activity in the supernatant of HIV-1-infected mitogen-stimulatedperipheral blood leukocytes at infected at 10 and 1.0 ng p24/10⁶ cells,FIGS. 3A and 3B, respectively.

[0042] FIGS. 4A-F. The structures of the compounds used in Example 7 areshown respectively in FIGS. 4A-4F. FIG. 4A:2-amino-4-(3,5-diacetylphenyl)amino-1,6-dimethylpyrimidinium chloride(CNI-0294). FIG. 4B:2-amino-4-(3,5-diacetylphenyl)amino-6-methylpyrimidine (CNI-1194). FIG.4C: 2-amino-4-(3-acetylphenyl)amino-6-methylpyrimidine (CNI-1594). FIG.4D: 2-amino-4-(4-acetylphenyl)amino-6-methylpyrimidine (CNI-1794). FIG.E: 3.5-diacetylaniline (CNI-1894). FIG. 4F:4-phenylamino-2-amino-6-methylpyrimidine (CNI-4594).

[0043]FIG. 5. Representative plasma concentrations over time in micetreated with CNI-1194. Female ND4 Swiss-Webster mice were given a single50 mg/kg injection intraperitoneally (circles) or orally (squares). Thecalculated plasma concentrations, in μg/ml, was then plotted against thetime of sampling.

[0044] FIGS. 6A-6B. Chromatograms of plasma extracts from animalstreated with CNI-0294 or CNI-1594. Female ND4 Swiss-Webster mice weregiven a single i.p. injection of 50 mg/kg CNI-0294 (A) or 20 mg/kgCNI-1594 (B). The chromatogram shown for CNI-0294 was from the 2 hr timepoint, and that for CNI-1594 for the 1 hr time point. The peaks labeled“2” and “15” are the parent peaks for CNI-0294 and CNI-1594respectively. The other peaks in the chromatogram represent possiblemetabolites (labeled “x”) and endogenous plasma peaks.

[0045] FIGS. 7A-7D. The in vitro metabolism of the CNI compounds. Thedrugs were incubated with mouse liver post-mitochondrial supernatantsand NADPH for various lengths of time. The chromatograms shown are fromthe 60 min time point for (A) CNI-0294, (B) CNI-1194, (C)CNI-1594, and(D) CNI-1894. The peaks labeled “2, 11, 15, 18” refer to the parentcompound peaks, and those labeled “an-n” to putative metabolite peaksthat increased over time and were not present in control incubations.All off-scale peaks were single peaks, and the scale was chosen to allowpresentation of trace metabolite peaks.

[0046] FIGS. 8A-8D. The in vivo metabolism of the CNI compounds. FemaleND4 Swiss Webster mice received a single intraperitoneal dose of (A) 50mg/kg CNI-0294, (B) 50 mg/kg CNI-1194, (C) 20 mg/kg CNI-1594, or (D) 50mg/kg CNI-1894. In all four graphs, the open bar represents the peakarea of the parent compound and the black bars the apparent metabolitepeaks. The metabolite peaks shown are (from left to right in eachgraph): (a) peak “d” (see FIG. 7 for letter-designated peaks), peak “a”,peak “c”, and a peak eluting at 13 minutes; (b) peak “h”, peak “e”, peak“f”, peak “g”, a peak eluting at 14 minutes, and a peak eluting at 23minutes; (C) peak “j”, peak “i”, peak “l”, and a peak eluting at 14minutes; (D) peak “m”, peak “n”, and a peak eluting at 11 minutes. Thepeak area units are arbitrary and calculated by the HPLC operatingsystem.

[0047]FIG. 9. The activity of CNI-0294 against Plasmodium bergheiinfected mice. Female ND4 Swiss Webster mice were infected with infectederythrocytes and then treated once daily, for four days, with 50 mg/kgCNI-0294, or with distilled water. Six hours after the last dose, thinblood smears were made from each of the animals and the parasitemia wasdetermined. The bars represent the median parasitemia (n=4 for controlsand n=5 for treated).

[0048] FIGS. 10A-10C. Binding of HIV-1 nucleoprotein complexes tokaryopherin α.

[0049] A. Binding of HIV-1 to Karyopherin α is Mediated by Both MA andVpr.

[0050] Cytoplasmic extracts prepared 4 hours after infection of H9 cellswith equivalent amounts (100 ng of p24 per 10⁶ cells) of wild-typeHIV-1_(NLHX) or variants carrying inactivating mutations in MA NLSor/and Vpr were divided in two aliquots. DNA was extracted from onealiquot and quantified by PCR using primers specific for the HIV-1 polgene (bottom panel). The obtained signal represented the total amount ofthe HIV-1 DNA in the cytoplasm. The second aliquot was incubated withGST-karyopherin α immobilized on Sepharose beads. HIV-1 DNA wasextracted from the beads and analyzed by PCR using pol-specific primers.The obtained signal represented the amount of HIV-1 pre-integrationcomplexes bound to karyopherin α.

[0051] B. Binding of HIV-1 Pseudovirion nucleoprotein complexes tokaryopherin α is mediated by MA NLS.

[0052] H9 cells were inoculated with HIV-like gag-env pseudovirions thatcontain gag RNA; equal amounts of wild-type (wt) and mutantpseudovirions that carry amino acid substitutions in the MA NLS (AMANLS) were used. Cytoplasmic extracts prepared from infected cells wereincubated with polyclonal anti-MA serum (ΔMA, lane 2), pre-immune serum(NSS, lane 3), or nothing (lanes 1, 4, and 5). Samples were then mixedwith GST-karyopherin α immobilized on glutathione Sepharose beads for 30min at 25° C. Nucleic acids were extracted from Sepharose beads andquantified by RT-PCR using primers specific for HIV-1 gag gene. Tocontrol for possible differences in cell entry of wild-type vs. mutantagents, HIV-specific nucleic acids were extracted directly fromcytoplasmic extracts and assayed by RT-PCR (lanes 4 and 5).

[0053] C. CNI-H0294 inhibits interactions of karyopherin α with HIV-1pre-integration complexes, but not with pseudovirion-derivednucleoprotein complexes.

[0054] Cytoplasmic lysates of H9 cells infected with HIV-1RF (upperpanel) or HIV-like pseudovirions (bottom panel) were treated for 2 hourswith various concentrations of CNI-H0294, and were then mixed withGST-karyopherin α immobilized on glutathione Sepharose beads. HIV-1 DNAor RNA that co-precipitated with karyopherin α was quantified as in Aand B.

[0055]FIG. 11. CNI-H0294 binds to recombinant RT in solution.

[0056] Twenty nmol of [¹⁴C]-CNI-H0294 were mixed with 0.28 nmol ofrecombinant MA or RT (a p51/p66 heterodimer) in 40 μl of binding buffer.Samples were incubated for 2 h at 37° C. in the presence or absence of200 nmol of unlabeled CNI-H0294. MA and RT proteins wereimmunoprecipitated using protein G agarose and sheep polyclonal anti-MA(αMA) or rabbit anti-RT (αRT) sera, respectively. Pre-immune sera (PI)was used as control. Bound material was eluted from protein G using 0.1M glycine buffer [pH 2.8] and the radioactivity in the eluate wasquantified in a scintillation counter.

[0057] FIGS. 12A-12C. CNI-H0294 interacts with RT to produce theanti-HIV effect.

[0058] A. CNI-H3094 competes with CNI-H0294 for binding to RT.

[0059] Recombinant RT (0.2 μM) was incubated with 3.3 μM of[¹⁴C]-labeled CNI-H0294 and increasing concentrations of unlabeled(cold) CNI-3094. The amount of [¹⁴C]-CNI-H0294 that bound to RT wasmeasured as in FIG. 11.

[0060] B. CNI-H3094 reduces CNI-H0294-mediated inhibition ofHIV-karyopherin α interaction.

[0061] Cytoplasmic lysates prepared from H9 cells infected with HIV-1RFwere treated with 10 μM CNI-H3094 (lane 1) or with 1 μM CNI-H0294 and 10μM (lane 5), 5 μM (lane 4), 1M (lane 3), or no CNI-H3094 (lane 2). Theamount of pre-integration complexes available for interaction withkaryopherin α was quantified as in FIG. 10.

[0062] C. CNI-H3094 inhibits anti-HIV activity of CNI-H0294 in monocytecultures.

[0063] Monocytes infected with HIV-1_(ADA) were cultured in the presenceof CNI-H0294 and CNI-H3094 in various concentrations. Nine days afterinfection, RT activity in culture supernatants was quantified. Theresults are presented as percent of total RT activity in untreatedcultures (control). Three independent samples were assayed for each drugconcentration, and the standard deviation was less than 15%.

[0064] FIGS. 13A-13B. Analysis of CNI-H0294 interactions with the HIV-1pre-integration complex.

[0065] A. CNI-H0294 does not disrupt the interaction between MA andHIV-1 cDNA.

[0066] Cytoplasmic extracts prepared from H9 cells infected with HIV-1RFwere treated with 10 μM CNI-H0294 (lanes 2, 3, and 4) or left untreated(lane 1). Samples were divided into two aliquots. One aliquot was mixedwith GST-karyopherin α immobilized on Sepharose beads and the HIV-1 DNAthat bound was quantified by PCR. The second aliquot wasimmunoprecipitated (IP) with anti-MA serum (αMA, lane 3) or withpre-immune serum (NSS, lane 4) as control.

[0067] B. CNI-H0294 reacts with MA, but only when MA is associated withthe HIV-1 pre-integration complex.

[0068] Cytoplasmic extracts from HIV-1RF-infected H9 cells were treatedwith [¹⁴C]CNI-H0294 (10 μM). After borohydride reduction the extractswere immunoprecipitated with anti-MA (αMA), anti-IN (αIN), or pre-immuneserum (PI). As control, similar reactions were performed using lysatesof pseudovirion-infected cells which lack RT and thus do not bindCNI-H0294. Immunoprecipitated radioactivity was quantified in ascintillation counter.

[0069] FIGS. 14A-14B. CNI-H0294 inhibits MA-, but not Vpr-mediatedbinding of HIV-1 pre-integration complexes to karyopherin α.

[0070] H9 cells were infected with equal amounts of wild-typeHIV-1_(NLHX) or with mutant HIV-1_(NLHX) that lack Vpr (Vpr⁻) or carry amutation that inactivates the MA NLS (MA NLS⁻). Infected cells werewashed and incubated for 4 h. An aliquot of each sample was used toquantify the total HIV-1 DNA (FIG. 14A) and the rest was used to preparecytoplasmic extracts. Extracts were incubated with 1 μM CNI-H0294 (FIG.14B, lanes 2, 4, 6, 8) or were left untreated (lanes 1, 3, 5, 7). Theamount of pre-integration complexes available for binding to karyopherinα was determined as in FIG. 10.

[0071]FIG. 15. Proposed mechanism of CNI-H0294 action.

[0072] CNI-H0294 binds to HIV-1 pre-integration complexes via RT, andthen reacts with an adjacent MA NLS. This interaction prevents bindingof karyopherin α to MA NLS; this nearly abolishes nuclear import of thepre-integration complexes. Note that Vpr binds to karyopherin α even inthe presence of CNI-H0294; this explains the low level of importdetected in the presence of the drug.

5. DETAILED DESCRIPTION OF THE INVENTION 5.1 THE COMPOUNDS AND METHODSOF THEIR SYNTHESIS

[0073] The present invention encompasses a class of alkyl aryl carbonylcompounds that forms stable, but preferably reversible, and mostpreferably reversible covalent interactions with one or more basic aminoacid residues, wherein such basic amino acid residues are a part of anuclear localization signal (NLS). The stable covalent interactionresults in the inhibition or neutralization of the nuclear localizationactivity of the NLS.

[0074] Two structural features are involved in conferring suchcapabilities of a compound of the invention: (a) a moiety comprising atleast one carbonyl group that reacts with the side chains of basic aminoacid residues in the target protein, i.e., the reactive group; and (b) asecond moiety, i.e., the targetting group, that interacts with aspecific docking site and determines the specificity of the compound fordifferent NLS.

[0075] Although the reactive group(s) of the compounds of the inventioncan react reversibly with any susceptible basic side chains in aprotein, e.g., arginines and lysines, the interaction between thetargetting group and the docking site confers specificity to theactivity of the carbonyl group(s), such that the carbonyl group(s) reactonly with particular target residue(s) in a protein. This targettingfunction occurs by interaction of the targetting group with a dockingsite that is located proximately to the susceptible side chains of basicamino acid residues of the target NLS, such that docking of the compoundplaces it in a favorable configuration to form a stable interaction withthe side chains of the basic amino acid residues of the target NLS. Itis to be understood that the docking site is located either on the sameNLS-bearing protein, or on another component of a larger molecularcomplex that includes the NLS-bearing protein.

[0076] Preferred compounds of the invention provide divalent arylcarbonyl moieties as the reactive group, particularly aryl bis(ketone)or aryl bis(α-diketone), aryl bis(β-diketone), linked to a targettinggroup, preferably to a nitrogen-containing heterocyclic functionalityvia an N-linkage. Particularly preferred compounds provided are bisacetyl, propanoyl, glyoxyloyl, pyruvoyl, 2-oxobutanoyl, acetoacetyl,3-oxopentanoyl, 3-oxo-2,2-dimethylbutanoyl or3-oxo-2,2-dimethylpentanoyl substituted aniline moieties N-linked to apyrimidinium, pyrimidine or triazine moiety.

[0077] The compounds of the present invention form reversible adductswith a target NLS containing protein. In particular, the compoundsform-Schiff bases with adjacent lysine residues, and other reversibleadducts with adjacent arginine residues. Thus, the compounds of theinvention, e.g., aryl bis(ketone), can advantageously be used forinactivation of an NLS where the NLS comprises lysine residues. On theother hand, where the NLS contains arginine residues, aryl bis(diketone)compounds, particularly dimethyl-substituted compounds, i.e., thoselacking a methylene hydrogen between the ketones, can be usedadvantageously.

[0078] In one particular embodiment, the compounds of the invention arecapable of forming Schiff bases with lysine residues of a target NLScontaining protein in a molecular complex, and interacting with aspecific docking site on a molecule in the molecular complex, saiddocking site being positioned proximately to the lysine residues in theprotein. Because of the proximity of the target lysine residues to thedocking site, the interaction of the targetting group with the dockingsite localizes the compound of the invention to the vicinity of thetarget lysine residues, thereby facilitating the reaction of thecarbonyl group(s) of the compound with the target N^(ε) group of lysineresidue(s) in the NLS, and resulting in the formation of stable Schiffbases and inactivation of the NLS.

[0079] Specific compounds of the invention, e.g., compound No. 2 orCNI-H0294, or2-Amino-4-(3,5-diacetylphenyl)amino-1,6-dimethylpyrimidinium salts, andtheir synthesis are described in section 6 et seq, and are disclosed inearlier filed U.S. patent application Ser. Nos. 08/369,830, 08/463,405,08/471,797, 08/470,103, and 08/584,857 each of which is herebyincorporated by reference.

[0080] According to the invention, the compounds of the invention arealkyl aryl carbonyl compounds of formula (I)

[0081] wherein A, independently, ═CH₃, CH₂CH₃, COH, COCH₃, COCH₂CH₃,CH₂COCH₃, CH₂COCH₂CH₃, C(CH₃)₂COCH₃, C(CH₃)₂COCH₂CH₃ or the like toyield an acetyl, propanoyl, glyoxyloyl, pyruvoyl, 2-oxobutanoyl,acetoacetyl, 3-oxopentanoyl, 3-oxo-2,2-dimethylbutanoyl or3-oxo-2,2-dimethylpentanoyl substituted aniline; P=1 or 2; L is a linkergroup containing an S, O, N or C atom, e.g., —SO₂—, —O—, —NH—, —N═,—CH₂— or —CH═; K is 0 or a positive integer, preferably K=1; and whereinJ represents (i) a saturated or unsaturated, substitued orunsubstituted, straight or branched acyclic hydrocarbon group; (ii) asaturated or unsaturated, substitued or unsubstituted, straight orbranched acyclic group containing hetero atoms such as nitrogen, sulfuror oxygen; (iii) a substituted or unsubstituted, saturated or aromatic,mono- or poly-cyclic group having 3 to 20 carbon atoms; or (iv) asubstituted or unsubstituted, saturated or aromatic, mono- orpoly-heterocyclic group having 3 to 20 atoms, at least one of which is anitrogen, sulfur or oxygen. The compounds of the invention can containone or more linker groups (L), however, if J contains a linker group asdefined above, K can be 0.

[0082] The acyclic and cyclic groups defined above may be saturated orunsaturated and may, if desired, bear one or more substituents.Exemplary of such substituents are alkyl, alkoxy, phenoxy, alkenyl,alkynyl, phenylalkyl, hydroxyalkyl, haloalkyl, aryl, arylalkyl,alkyloxy, alkylthio, alkenylthio, phenylalkylthio, hydroxyalkyl-thio,alkylthiocarbamylthio, phenyl, cyclohexyl, pyridyl, piperidinyl,alkylamino, amino, nitro, mercapto, cyano, hydroxyl or a halogen atom.

[0083] For example, J can be a substituted or unsubstituted five or sixmembered ring having 1-4 hetero ring atoms, at least one of which isnitrogen, the remainder of which are selected from nitrogen, oxygen orsulfur, e.g., pyridine, pyrrole, imidazole, thiazole, isothiazole,isoxazole, furazan, pyrrolidine, piperidine, imidazolidine, piperazine,oxazole, tetrazole, pyrazole, triazole, oxadiazole, thiodiazole.Alternatively, J can be a substituted or unsubstituted polycyclic grouphaving 1 to 4 hetero ring atoms, one of which is nitrogen and theremainder of which are nitrogen, oxygen or sulfur, e.g., indole,quinoxaline, quinazoline, quinoline, isoquinoline, purine.

[0084] By the term “alkyl” as used herein is meant a straight orbranched chain saturated hydrocarbon group having from 1 to 20 carbonssuch as methyl, ethyl, isopropyl, n-butyl, s-butyl, t-butyl, n-amyl,isoamyl, n-hexyl, n-octyl and n-decyl. The terms “alkenyl” and “alkynyl”are used to mean straight or branched chain hydrocarbon groups havingfrom 2 to 10 carbons and unsaturated by a double or triple bondrespectively, such as vinyl, allyl, propargyl, 1-methylvinyl,but-1-enyl, but-2-enyl, but-2-ynyl, 1 methylbut-2-enyl, pent-1-enyl,pent-3-enyl, 3-methylbut-1-ynyl, 1,1-dimethylallyl, hex-2-enyl and1-methyl-1-ethylallyl. The term “phenylalkyl” means the aforementionedalkyl groups substituted by a phenyl group such as benzyl, phenethyl,phenopropyl, 1-benzylethyl, phenobutyl and 2-benzylpropyl. The term“aryl” as used herein is meant to include a monocyclic, bicyclic,tricyclic or other polycyclic compounds, wherein at least one ring isaromatic including aromatic hydrocarbons or hetero-aromatic hydrocarbonshaving heteroaromatic atoms such as nitrogen, sulfur and oxygen. Theterm “hydroxy-alkyl” means the aforementioned alkyl groups substitutedby a single hydroxyl group such as 2-hydroxyethyl, 2-hydroxypropyl,3-hydroxypropyl, 4-hydroxybutyl, 1-hydroxybutyl and 6-hydroxyhexyl. Theterms “alkylthio, alkenylthio, alkynylthio, hydroxy-alkylthio andphenyl-alkylthio” as used herein mean the aforementioned alkyl, alkenyl,alkynyl, hydroxy-alkyl and phenyl-alkyl groups is linked through asulfur atom.

[0085] The term “substituted” as used herein means that the group inquestion, e.g., alkyl group, aryl group, etc., may bear one or moresubstituents including but not limited to halogen, hydroxy, cyano amino,nitro, mercapto, carboxy and other substituents known to those skilledin the art.

[0086] The terms “saturated” as used herein means an organic compoundwith neither double or triple bonds. The term “unsaturated” as usedherein means an organic compound containing either double or triplebonds.

[0087] In another embodiment, the compounds of the invention are formedaccording to formula (II):

[0088] wherein A, independently, ═CH₃ or CH₂CH₃ and P=1 or 2; and

[0089] wherein X═NH₂, CH₃ or CH₂CH₃; X′=CH₃ or CH₂CH₃; Y═NH₂, NHCH₃,N(CH₃)₂, 1-pyrrolidino or 1-piperidino; and Z=H, CH₃ or CH₂CH₃; or

[0090] wherein Y′ and Z′, independently, ═H, NH₂, NHCH₃, N(CH₃)₂ or N⁺(CH₃)₃, 1-pyrrolidino or 1-piperidino; Q is N or CH; and salts thereof.

[0091] In a preferred embodiment the compounds of the invention are bisketone arylene compounds having a third nitrogenous substituent. Thenitrogenous substituent can be further substituted with an aromaticnitrogen-containing heterocyclic compound.

[0092] More precisely the bis ketone arylene compounds of the inventionare formed according to the formula (III):

[0093] wherein A=CH₃ or CH₂CH₃ and

[0094] wherein X═NH₂, CH₃ or CH₂CH₃; X′═CH₃ or CH₂CH₃; Y═NH₂, NHCH₃,N(CH₃)₂, 1-pyrrolidino or 1-piperidino; and Z=H, CH₃ or CH₂CH₃; or

[0095] wherein Y′ and Z′, independently, ═H, NH₂, NHCH₃, N(CH₃)₂ orN⁺(CH₃)₃, 1-pyrrolidino or 1-piperidino; Q is N or CH; and saltsthereof.

[0096] In yet another embodiment of the invention, the compounds of theinvention are compounds formed according to formula (I), wherein J isnot R; L is not —NH—; and wherein

[0097] and wherein, independently, X═NH₂, CH₃ or CH₂CH₃; X′═CH₃ orCH₂CH₃; Y═NH₂, NHCH₃, N(CH₃)₂; and Z=H, CH₃ or CH₂CH₃; or

[0098] wherein Y′ and Z′, independently, ═H, NH₂, NHCH₃, N(CH₃)₂ orN⁺(CH₃) 3; and salts thereof.

[0099] The compounds of the present invention can be synthesized byreacting aniline—to form a compound of formula (II), wherein P is 0—oran acetyl or propanoyl derivative of aniline—to form a compound offormula (II), wherein P is 1—or a diacetyl or dipropanoyl derivative ofaniline—to form a compound of formula (II) or formula (III) wherein P is2—with a chloro derivative of purine, aminomethylpyrimidine,diamino-triazine, or with a cyanoguanidine. The reaction can beperformed at 90-100° C. in an aqueous solvent in the presence of amineral acid to yield the corresponding aminophenyl pyridine ortriazine. The pyrimidinium can be synthesized from the pyrimidine byreaction with an excess methyl iodide at 40-45° C. under refluxconditions in 1:1 acetonitrile/tetrahydrofuran or in a 1:1:2 mixture ofdichloromethane/acetonitrile/tetrahydrofuran.

[0100] For synthesis of aryl bis(diketone) compounds, the acyl groupsattached to the benzene ring, for example in 3,5-diacetylaniline, may beconverted to 2-oxoacyl groups by reaction with selenium dioxide orselenious acid in wet dioxane or other suitable medium (Rabjohn, N.,1976, Org. React. 24:261-415). Acyl groups attached to benzene rings maybe converted to higher-chain 3-oxoacyl groups by treatment with strongbase such as sodium hydride or sodium metal in the presence of analiphatic ester such as ethyl acetate, when the acyl groups are of thestructure X—CH₂—CO— (March, J., 1992, Advanced Organic Chemistry, 4thed., Wiley Interscience, New York, pp491-493; Fenton, D. E. et al.,1980, Inorg. Chim. Acta 44:L105-L106)

[0101] Any chemistries for generating chemical libraries known in theart can be used to form the J group in the compound of formula (I),including but not limited to combinatorial chemistries, in whichinterchangeable chemical building blocks are systematically assembled toprovide diverse structures. See generally Gordon, E. M. et al., 1994, J.Med. Chem. 37:1385; Chen, C. et al., 1994, J. Amer. Chem. Soc. 116:2661;Cho, C. Y. et al., 1993, Science 261:1303. The modification andadaptation of the various chemistries for generating diversities for theformation of the J group in the compound of formula (I) will be apparentto persons of skill in the art. Moreover, automated synthesis systemscan be used to generate the desired chemical diversities including, forexample, workstations and robots made by Takeda Chemical Industries Ltd,Osaka, Japan; Zymark Corporation, Hopkinton, Mass.; and Hewlett Packard,Palo Alto, Calif.

5.2 THE INHIBITION OF HIV-1 IMPORTATION INTO THE NUCLEUS OF NON-DIVIDINGCELLS

[0102] A quantitative measurement of the activity of the compounds ofthe invention to block the replication of HIV-1 in non-dividing cellscan be determined by culture of a macrophage-tropic strain of HIV-1 onperipheral blood-derived macrophages. The cells are cultured for 5-6days prior to infection in a medium consisting of DMEM supplemented with10% type A/B human serum and 200 U/ml Macrophage Colony StimulatingFactor, with half the medium changed after 3 days, to reach a density ofabout 10⁶ cells per 5 ml well. A macrophage-tropic viral stock may begrown on these cells. The concentration of infectious particles in thestock is estimated by measurement of p24 antigen concentration. To testthe effect of compounds of the invention on HIV-1 infection in theabove-described culture system, the medium is removed and replaced withmedium containing HIV-1 at a concentration of 1 ng of p24 (10⁴ TCID₅₀/ml(TCID=tissue culture infectious doses)) and a known concentration of thecompound of the invention (the inhibitor). After 24 hours, the culturesare washed to remove non-adherent virus and the culture is re-fed withmedium containing the inhibitor at the desired concentration. The amountof replication of HIV-1 is estimated by an assay of the reversetranscriptase activity or by an assay of the concentration of p24antigen in the culture medium every 2-3 days throughout thepost-infection period. In a preferred embodiment the anti-HIV potency ofthe candidate drug is measured by comparison of the concentration ofreverse transcriptase (RT) or of p24 antigen in the medium of thetreated and control cultures at the time of the peak of these values innon-treated control cultures, that is about day 5 or 6 post-infection.Repetition at various levels of inhibitor allows for the calculation ofthe concentration of inhibitor that achieves 50% inhibition of viralgrowth, IC₅₀. Table I discloses the IC₅₀ of various inhibitors. TABLE ICompound IC₅₀ 2-amino-4-(3,5-diacetylphenyl)amino-1,6-  1 nMdimethylpyrimidinium iodide (Compound No. 2)2-amino-4-(3-acetylphenyl)amino-1,6- 10 nM dimethylpyrimidinium iodide(Compound No. 14) 2-amino-4-(3,5-diacetylphenyl)amino-6- 50 nMmethylpyrimidine (Compound No. 11) 4-(3-acetylphenyl)amino-2-amino-6- 15nM methylpyrimidine (Compound No. 15)

[0103] Alternatively, the compounds may all be compared for inhibitionof HIV replication at a fixed concentration. Presented in Table II arecompounds that were used at a concentration of 100 nM to inhibit theproduction of HIV-1 in cultured monocytes infected with HIV-1 10 daysprior to assay (10 ng of p24/10⁶ cells). The production of HIV-1 in eachtreated culture is reported as percentage of untreated control. TABLE IIViral Compound Production N-(3,5-diacetylphenyl)biguanide hydrochloride12% (Compound No. 12)2-(3,5-diacetylphenyl)amino-4,6-diamino-1,3,5-triazine 14% (Compound No.13) 4-(3-acetylphenyl)amino-2-amino-6-methylpyrimidine 20% (Compound No.17) 3,5-diacetylaniline 20% N,N-dimethyl-3,5-diacetylaniline 25%2,6-diacetylaniline 28% 3,5-diacetylpyridine 58%

[0104]FIG. 2A presents further results of the use of the most active ofthe compounds of Table I, Compound No. 2, to block the replication ofHIV-1 in purified monocytes, cultured in medium supplemented withmonocyte-colony stimulating factor (M-CSF). The cultures were treatedwith none or between 10⁻¹² and 10⁻⁶ M Compound No. 2 and, simultaneouslywith the beginning of treatment, the cells were exposed to themonocyte-tropic strain HIV-1_(ADA) at about 0.01 TCID₅₀/cell (1 ngp24/10⁶ cells) for 2 hours. Samples were withdrawn at days 3, 6, 10, 14and 17 after infection and assayed for reverse transcription activity.Compound No. 2 does not inhibit reverse transcriptase, data not shown.The results show that under these conditions the IC₅₀ concentrations isbetween 0.1 and 1.0 nM and that a concentration of between 0.1 μM and1.0 μM completely inhibits the replication of the virus.

[0105]FIGS. 2B and 2C show the effects of various concentrations ofCompound No. 2 on the production of HIV-1 in monocyte cultures notsupplemented with M-CSF. In these studies MOI, as determined byconcentration of p24 antigen was; FIG. 2B (8 ng/10⁶ cells) and FIG. 2C(0.8 ng/10⁶ cells). These experiments showed IC₅₀s of about 10 nM and ofless than 1.0 nM respectively.

[0106] The inhibition of the replication of HIV-1 is not due to generalcytotoxic effects of the compound. Concentrations of Compound No. 2 ashigh as 10 μM were without toxic effects on the monocyte cultures asdetermined by lactate dehydrogenase release and trypan blue exclusion.Further evidence of the specificity of the inhibition due to CompoundNo. 2 is provided by the data presented in FIGS. 3A and 3B whereinmitogen-stimulated peripheral blood leukocytes were cultured inIL-2-supplemented medium and were exposed to the HIV-1_(ADA) at p24concentrations of 10 and 1 ng/10⁶ cells, respectively. In thisexperiment up to 10 μM Compound No. 2 had only a marginal effect onviral production at the higher MOI. At the lower MOI, 1 and 10 μM ofCompound No. 2 caused an approximate 2-fold reduction in viral output.

[0107] The inhibition of HIV-1 importation into the nucleus ofnon-dividing cells can also be directly measured. One suitable method todetermine directly the activity of compounds of the invention utilizes acell line that is susceptible to HIV-1 infection, e.g., MT-4 cells, thatis growth arrested by treatment with aphidicolin and exposed to HIV-1.PCR amplification is used to detect double-stranded closed circularHIV-1 genomes, which are formed only after nuclear importation, byselecting primers that bridge the junction point of the genome. Forgreater detail see Bukrinsky, M. I., et al., 1992, Proc. Natl. Acad.Sci. 89:6580-84.

5.3 THE TREATMENT OF HIV INFECTION

[0108] The present invention provides a method of treatment of HIV-1infection by administering to an HIV-1-infected subject a pharmaceuticalcomposition having, as an active ingredient, an effective amount of acompound of formula (I) and (III), and particularly a compound offormula (III). In one embodiment the compound to be administered isCompound No. 2. Pharmaceutical compositions suitable for oral,intraperitoneal, and intravenous administration can be used in thepractice of the invention. Such pharmaceutical compositions include, byway of non-limiting examples, aqueous solutions of the chloride,bicarbonate, phosphate and acetate salts of Compound No. 2 andpH-buffered mixtures thereof. The chloride salt of compound 2 is hereinreferred to as CNI-0294. Compound 11, Compound 14 and Compound 15 arealso known as CNI-1194, CNI-H1494 and CNI-1594, respectively.

[0109] The effective dose of the active ingredient can be determined bymethods well known to those skilled in medicinal chemistry andpharmacology. An effective dose is the dose that achieves in thesubject's plasma a concentration of the active ingredient that issufficient to inhibit the replication of HIV-1 in monocyte cultures asdescribed in Section 5.4, supra, but does not lead to cytopathic effectsin such cultures.

[0110] The daily dose and dosing schedule to be given a subject can bedetermined by those skilled in the art, using the pharmacokineticconstants set forth in Table III below, to achieve a target plasmaconcentration. The target plasma concentration can be selected byroutine pharmacological and clinical investigation methods well-known tothose skilled in the art, and can be based on a range of concentrationswhich encompass the IC₅₀ calculated for each particular compound. Forexample, the dose can be adjusted to achieve a range of target plasmaconcentrations that included the IC₅₀ for the compounds as shown inTable I above. TABLE III Pharmacokinetic parameters of the CNIcompounds. CNI- CNI- CNI- CNI- CNI- CNI- CNI- 0294 0294 0294 1194 11941594 1894 Route of i.p. i.p. oral i.p. oral i.p. i.p. Injection Dose(mg/kg) 50 50 50 50 50 20 50 Vehicle DP* W* DP W W W W AUC (μ * hr/ml)9.15 8.83 0.56 3.93 0.57 0.82 20.20 C_(max) (μg/ml) 18.76 18.93 0.415.70 0.35 1.93 13.43 t_(max) (min) 5 5 60 15 15 15 5 α (hr⁻¹) 1.12 1.74— 1.83 — 2.14 1.19 β (hr⁻¹) 0.15 0.19 — 0.19 — 0.04 0.03 A (μg/ml) 14.0016.07 — 5.22 — 1.10 14.93 B (μg/ml) 0.07 0.05 — 0.14 — 0.01 0.15t_(1/2α) (hr) 0.62 0.40 — 0.38 — 0.32 0.58 t_(1/2β) (hr) 4.62 3.65 —3.65 — 17.33 23.10 V_(D) (L) 14.14 19.80 — 5.21 — 39.60 6.60 Cl_(tot)(ml/min) 35.35 62.70 — 16.50 — 26.40 3.30 Bioavailability — — 0.06 —0.15 — —

[0111] For example, using the foregoing pharmacokinetic constants,particularly, the clearance rate, the daily dose and dosing scheduleneeded to obtain a given target average plasma concentration can becalculated. The results of such calculations for Compound Nos. 2, 11 and15 are presented in Table IV. The calculated doses of Compound Nos. 2and 15 are considerably below the toxic levels, as measured by the LD₅₀,of these compounds. See, Section 6.4 below. TABLE IV Compound TargetClearance‡ Dose No. M.W. serum conc. (ml/min) (mg/Kg day)  2* 334 10 nM35.35  6.80 11 280 50 nM 16.50 13.3  15 250 15 nM 26.40  5.70

[0112] Using such methods, a dose can be calculated to achieve apredetermined target plasma concentration. A practicable target plasmaconcentration of Compound No. 2 ranges from 0.5 nM to 10 nM; forCompound No. 11, a practicable target range is from 25 nM to 100 nM; forCompound No. 15, a practicable target range is from 7.5 nM to 50 nM.

[0113] Subjects who can benefit from the administration of the compoundsof the invention according to this method include all persons infectedby HIV-1. More particularly, firstly, those who benefit include thosesubjects who have or are at risk to develop CNS signs of HIV-1 infectionand/or subjects that have developed significant weight loss. Secondly,those who benefit include those who have been recently exposed to HIV-1,but who do not yet have an established chronic infection.

5.4 PHARMACEUTICAL FORMULATIONS

[0114] Because of their pharmacological properties, the compounds of thepresent invention can be used especially as agents to treat patientssuffering from HIV and can be used as agents to treat patients sufferingfrom other viral infections or chronic diseases that are dependent uponnuclear localization as part of the pathogenic process. The compounds ofthe invention can also be used to treat or prevent other infectiousdiseases such as parasitic diseases, and in particular malaria. Such acompound can be administered to a patient either by itself, or inpharmaceutical compositions where it is mixed with suitable carriers orexcipient(s).

[0115] Use of pharmaceutically acceptable carriers to formulate thecompounds herein disclosed for the practice of the invention intodosages suitable for systemic administration is within the scope of theinvention. With proper choice of carrier and suitable manufacturingpractice, the compositions of the present invention, in particular,those formulated as solutions, may be administered parenterally, such asby intravenous injection. The compounds can be formulated readily usingpharmaceutically acceptable carriers well-known in the art into dosagessuitable for oral administration. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya patient to be treated.

[0116] Pharmaceutical compositions suitable for use in the presentinvention include compositions wherein the active ingredients arecontained in an effective amount to achieve its intended purpose.Determination of the effective amounts is well within the capability ofthose skilled in the art, especially in light of the detailed disclosureprovided herein.

[0117] In addition to the active ingredients these pharmaceuticalcompositions may contain suitable pharmaceutically acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.The preparations formulated for oral administration may be in the formof tablets, dragees, capsules, or solutions.

[0118] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is itself known, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levitating,emulsifying, encapsulating, entrapping or lyophilizing processes.

[0119] Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

[0120] Pharmaceutical preparations for oral use can be obtained bycombining the active compounds with solid excipient, optionally grindinga resulting mixture, and processing the mixture of granules, afteradding suitable auxiliaries, if desired, to obtain tablets or drageecores. Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

[0121] Dragee cores are provided with suitable coatings. For thispurpose, concentrated sugar solutions may be used, which may optionallycontain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel,polyethylene glycol, and/or titanium dioxide, lacquer solutions, andsuitable organic solvents or solvent mixtures. Dyestuffs or pigments maybe added to the tablets or dragee coatings for identification or tocharacterize different combinations of active compound doses.

[0122] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added.

5.5 USE OF THE COMPOUNDS OF THE INVENTION TO DERIVATIZE PROTEINS

[0123] The compounds of the present invention of formula (III), whereinP is 1 or 2, can be used to derivatize a target protein and therebydetermine the presence of adjacent N^(ε)-moieties. The test reaction canbe conducted in aqueous buffer at mild to moderate alkaline pH, betweenabout 7.2 and 8.0. Specific derivatization of the target protein can bedetected by any means that separates protein-bound and free derivatizingcompound. The derivatizing compound optionally can be detected byradiolabeling it. In one embodiment, the compound can be synthesizedusing ¹⁴C-methyliodide in place of methyliodide. Alternatively, use canbe made of the strong UV absorption or fluorescence of the derivatizingcompounds. Compound No. 2, for example has a absorption peak of 16,000M⁻¹ cm⁻¹ at λ=298 nm. In a preferred embodiment the target protein isderivatized by a compound of the invention, irreversibly reduced withsodium borohydride or cyanoborohydride and fragmented into peptides bytrypsin or the like. The resultant peptides can be compared with thepeptides obtained from an unreacted sample of the protein by analysisusing any chromatographic or electrophoretic technique that resolvespeptides, e.g., reverse phase High Performance Liquid Chromatography(HPLC). When the peptides are resolved by any high resolutionchromatography procedure, the derivatized peptides can be readilydetected by their altered elution time and the absorbance at λ=298 nm.

[0124] In a preferred embodiment the practitioner will conduct thereaction at various pH points to determine whether a positive result canbe obtained at any point within the expected range. A positive result,i.e., a result that indicates the presence of adjacent N^(ε)-moieties,is one in which a large fraction of each of a limited number, i.e.,between 1-4, of peptides of the target protein are derivatized andnegligible amounts of other peptides are affected.

[0125] The above-described protein derivatization technique can be usedto determine whether a candidate compound can be used, according to theinvention to prevent productive HIV-1 infection of macrophages. Acomparison of the activity of a candidate compound and that of CompoundNo. 2 as derivatizing agents specific for nuclear localization sequencescan be made. A compound that derivatizes the same peptides to the sameextent as Compound No. 2 can be used to practice the invention.

5.6 THE TREATMENT OF INFECTIOUS DISEASES

[0126] The compounds of the present invention can be used to prevent ortreat infectious diseases in animals, including mammals and preferablyhumans, and these compounds are particularly suited to treatment ofparasitic diseases, more particularly, malaria. The invention describedherein provides methods for treatment of infection, including andwithout limitation, infection with parasites, and methods of preventingdiseases associated with such infection. The compounds can reduceparasitemia when administered to an animal infected with a parasite.

[0127] Infectious diseases may include without limitation: protozoaldiseases such as those caused by Kinetoplastida such as Trypanosoma andLeishmania, by Diplomonadina such as Giardia, by Trichomonadida such asDientamoeba and Trichomonas, by Gymnamoebia such as Naegleria and theAmoebida such as Entamoeba and Acanthamoeba, by Sporozoasida such asBabesia and the Coccidiasina such as Isospora, Toxoplasma,Cryptosporidium, Eimeria, Thelleria, and Plasmodium; metazoal diseasessuch as those caused by the Nematoda (roundworms) such as Ascaris,Toxocara, the hookworms, Strongyloides, the whipworms, the pinworms,Dracunculus, Trichinella, and the filarial worms, and by thePlatyhelminthes (flatworms) such as the Trematoda such as Schistosoma,the blood flukes, liver flukes, intestinal flukes, and lung flukes, andthe Cestoda such as the tapeworms; viral and chlamydial diseasesincluding for instance those caused by the Poxyiridae, Iridoviridae,Herpesviridae, Adenoviridae, Papovaviridae, Hepadnaviridae,Parvoviridae, Reoviridae, Birnaviridae, Togaviridae, Coronaviridae,Paramyxoviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae,Bunyaviridae, Arenaviridae, Retroviridae, Picornaviridae, Calciviridaeand by Chlamydia; bacterial diseases; mycobacterial diseases;spirochetal diseases; rickettsial diseases; and fungal diseases.

[0128] In one embodiment, the compounds of the invention havinganti-infective activity are formed according to formula (I), (II) and(III) as described in section 5.1.

[0129] In another embodiment, the compounds of the invention may be usedtherapeutically against infections with Plasmodium species such as P.falciparum, P. vivax, P. ovale and P. malariae, that cause acute andrecurrent malaria in humans. The compounds of the invention are alsoactive against infection by other Plasmodium species, which include P.berghei, P. knowlesi, P. simium, P. cynomolgi bastianelli and P.brasilianum.

[0130] In yet another embodiment of the invention, the compounds may beuseful in providing chemoprophylaxis for individuals at risk ofinfection, such as when travelling in endemic areas. By maintaining incirculation an effective concentration of a compound of the invention,malaria can be prevented by suppressing the pathological stages ofinfection with Plasmodium species. Without being bound by any theory,the compounds of the invention can be effective against various stagesof the life cycle of the parasite, including sporozoites and merozoites,as well as dormant, asexual and sexual stages. The compounds of theinvention may be active in the blood stream, in erythrocytes, in theliver, or in other tissues where the malaria parasite may reside.

[0131] In a specific embodiment of the invention, the compound of theinvention can be used to prevent malaria, or to treat malaria, or totreat infection with Plasmodium species that are resistant toantimalarial drugs, such as, but not limited to, chloroquine andpyrimethamine. The antimalarial properties of the compounds are notdiminished against P. falciparum known to be resistant to chloroquine orpyrimethamine (see section 8 infra). Although not wishing to be bound byany theory of mechanism of the compounds, it is contemplated that thecompounds interact with biochemical targets that are different andindependent from those affected by these two classic antimalarial drugs.Thus, the compounds of the invention may be used preferentially to treatmalarial infections arising out of areas that are known or suspected toharbor drug-resistant Plasmodium species.

[0132] In a further embodiment, the compounds may contain a single acylgroup, i.e., P=1, on the arylene ring or the acyl group can be absenttherefrom, i.e., P=0, and/or the heterocyclic substituent, i.e., R, canbe uncharged. In the embodiment of the invention wherein there are twoacyl groups, i.e., P=2, on the arylene ring, it is preferred that suchacyl groups are not in an ortho arrangement relative to each other. Inanother preferred embodiment of the invention, the compounds thatpossess potent antimalarial activity are arylene bis(methylketone)compounds that contain a charged heterocylic ring such as apyrimidinium, as in CNI-0294 (see FIG. 4A).

[0133] The antimalarial properties of the compounds of the invention canbe analyzed by techniques, assays and experimental animal models wellknown in the art. For example, the inhibition of growth of Plasmodiumfalciparum in vitro by the compounds may be assessed by thehypoxanthine-incorporation method (Desjardins et al., 1979, Antimicrob.Ag. Chemother. 16:710-718). The in vitro antiparasitic activities ofseveral exemplary compounds of the invention were assessed by thismethod, and the results are described in Section 8. The in vivo efficacyof the compounds can also be tested in mouse models in which parasitemiais enumerated following administration of the compound (Ager, A. L.1984, Rodent malaria models, pp 225-264. In Handbook of ExperimentalPharmacology vol. 68, Antimalarial Drugs, Peters and Richards eds,Springer-Verlag, Berlin). The in vivo activity of several exemplarycompounds have been evaluated in a four-day suppression model in mouse,and the results are provided in Section 8.

[0134] The present invention also provides pharmaceutical compositions.Such pharmaceutical compositions comprises a prophylactically ortherapeutically effective amount of the compound and a pharmaceuticalcarrier, such as those described in section 5.4. More specifically, aneffective amount means an amount effective to prevent development of orto alleviate the existing symptoms of the subject being treated.Determination of the effective amounts is well within the capability ofthose skilled in the art, especially in light of the detailed disclosureprovided herein. For any compound used in the method of the invention,the effective dose can be estimated initially from in vitro assays. Adose can be formulated in animal models to achieve a circulating rangethat includes the IC₅₀ (i.e., the concentration of compound whichachieves a half-maximal inhibition of growth of parasite) as determinedin the in vitro assay. Such information can be used to more accuratelydetermine useful doses in subjects, for example, humans. The dosage mayvary within this range depending upon the dosage form employed and theroute of administration. Various delivery systems are known and can beused for administration of the compound, e.g., encapsulation inliposomes. Other methods of administration include but are not limitedto intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal and oral routes.

[0135] In another embodiment, the invention provides a method ofpreventing or treating malaria by administering to a subject in needthereof an effective amount of a compound of the invention. In a furtheraspect there is provided a method of preventing or treating malaria,especially malaria caused by drug resistant Plasmodium species inhumans, which method comprise administering to the individual in needthereof an effective amount of a compound of the present invention andan effective amount of an antimalarial drug. The invention also providesthe use of a compound of the invention and an antimalarial drug in themanufacture of a medicament for the prevention or treatment of malaria.Such antimalarial drugs may include but are not limited to quinine,aminoquinolines (chloroquine and primaquine), pyrimethamine, mefloquine,halofantrine, and artemisinins.

[0136] The “adjunct administration” of a compound of the invention andan antimalarial drug means that the two are administered either as amixture or sequentially. When administered sequentially, the compoundmay be administered before or after the antimalarial drug, so long asthe first administered agent is still providing antimalarial activity inthe animal when the second agent is administered. Any of theabove-described modes of administration may be used in combination todeliver the compound and the antimalarial drug.

[0137] The present invention is to be understood as embracing all suchregimens and the term “adjunct administration” is to be interpretedaccordingly. When a compound of the invention and an antimalarial drugare administered adjunctively as a mixture, they are preferably given inthe form of a pharmaceutical composition comprising both agents. Thus,in a further embodiment of the invention, it is provided apharmaceutical composition comprising a compound of the invention and anantimalarial drug, together with a pharmaceutically acceptable carrier.

5.7 COMPOUNDS AND ASSAYS FOR COMPOUNDS THAT TARGET SPECIFIC NLS

[0138] The present invention also provides methods of use of the alkylaryl carbonyl compounds as described in section 5.1. The reactivegroup(s) of the compounds of the invention are capable of forming stablebut reversible covalent interactions with the side chain of basic aminoacid residue(s) of a protein. In certain embodiments of the invention,the divalency of the compound ensures that the compound will formespecially stable associations with sequences, such as nuclearlocalization signals (NLS), that comprise multiple basic amino acidresidues, such as lysines and arginines. Nuclear localization signals,in general, and the HIV matrix antigen (MA) p17 NLS in particular arecharacterized by a stretch of basic, and often positively charged, aminoacids typically including one or more lysines. Such NLS-containingproteins are often associated with other molecules in a complex.

[0139] The interaction of basic side chain(s) with the reactive carbonylgroup(s) of the compound is facilitated by the prior interaction of thetargetting group of the compound with a specific docking site, saiddocking site being positioned proximately to the side chain of the basicamino acid residue(s) in the target NLS. The docking site may be locatedon the NLS-containing protein or another molecule in a complexcomprising the NLS-containing protein. As a result of the formation ofstable but reversible covalent interactions with the compound, theNLS-containing protein or the molecular complex comprisingNLS-containing protein is prevented from interacting with cellularreceptors for the NLS. The protein or molecular complex is thus blockedfrom importation into the nucleus.

[0140] Although the carbonyl group(s) of the compounds of the inventioncan react with alternative susceptible side chains in a protein, theinteraction between the targetting group and the docking site confersspecificity to the activity of the carbonyl group(s), such that thecarbonyl group(s) react only with particular target basic amino acidresidues in a protein. The specific recognition and binding of thecompound to a docking site determines which basic amino acid residue(s)in a protein will become inactivated by the carbonyl group(s) of thecompound. Because of the proximity of the basic residues in the targetNLS to the docking site, the interaction of the compound with thedocking site localizes the compound to the vicinity of the target NLS,thereby facilitating the reaction of the carbonyl group(s) of thecompound with the target NLS. It is to be understood that the dockingsite can be on the NLS-containing protein or another molecule in acomplex comprising the NLS-containing protein. Thus, in one embodiment,the compounds of the invention can be used to target a specific nuclearlocalization signal, thereby blocking importation of specific proteinsor molecular complexes into the nucleus.

[0141] As demonstrated by the experiments in section 9, the compounds ofthe invention, particularly Compound No. 2 or CNI-H0294, are capable ofinhibiting nuclear translocation of HIV-1 pre-integration complexes. Theinhibitory effect is caused by the inactivation of the NLS of HIV MA ina reaction that requires the presence of HIV reverse transcriptase (RT),i.e. the target nucleoprotein complex. The carbonyl groups of CompoundNo. 2 or CNI-H0294 react with the NLS of MA by formation of Schiff baseadducts, and prevent the binding of MA to karyopherin α, the cellularreceptor for NLS, and that blocks translocation of proteins into thenucleus. The inventors further showed that the interaction betweenCompound No. 2 or CNI-H0294 via its pyrimidine moiety with RT thatcontains a docking site determines the specificity of the compoundtowards HIV pre-integration complex, and its low cytotoxicity to thehost cell. Thus, the alkyl aryl carbonyl compounds of the invention canbe used for targeted inactivation of NLS in a protein or a molecularcomplex, such as the HIV-1 or other viral pre-integration complex,comprising contacting the protein or molecular complex with a compoundof the invention. Furthermore, variations and modifications of thetargetting group provides for altered binding specificities, and canserve to target the alkyl aryl carbonyl compound to a differentNLS-containing protein or molecular complex. Thus, it is envisioned thatthe alkyl aryl carbonyl compounds of the invention, can be selected toinactivate specifically various different NLS-containing proteins ormolecular complexes.

[0142] Accordingly, it is contemplated that a molecular complexcomprises at least one NLS-containing protein and can include otherproteins, nucleic acids, carbohydrates, lipids and other biologicalmolecules. The docking site to which the targeting group of the compoundbinds may reside on any molecule in the molecular complex. In apreferred embodiment, the target of the compound of the presentinvention is a viral nucleoprotein complex or a viral protein.Alternatively, a target may be a cellular protein such as atranscription factor. An exemplary, non-limiting list of such targetsmay include, viruses which translocate both viral proteins and nucleicacids into the infected cell nucleus, such as human immunodeficiencyvirus and other retroviruses, influenza virus, hepatitis B and hepatitisdelta virus, papillomaviruses and parvoviruses; viruses whichtranslocate only viral protein into the infected cell nucleus such asadenoviruses, measles and other paramyxoviruses, herpes viruses, andrabies and other bunyaviruses; and single protein, such as NF-KB andother transcription factors.

[0143] In another embodiment of the invention, screening assays areprovided for identification of alkyl aryl carbonyl compounds of theinvention that can inactivate the NLS of a specific NLS-containingprotein or NLS-containing molecular complex. The assays of the inventioninvolves monitoring the binding of the NLS-containing protein ormolecular complexes to at least one protein or fragment thereof thatinteracts with the NLS in the course of nuclear translocation, i.e., thecellular receptor, in the presence of an alkyl aryl carbonyl testcompound of the present invention, and a comparison of such binding inthe absence of the test compound. It is anticipated that the desiredcompound binds specifically to a docking site in the NLS-containingprotein or molecular complex, and inactivates the NLS. As a result, thebinding of the NLS protein or molecular complex to the cellular receptoris reduced or abolished.

[0144] Any cellular protein(s) that interact with the NLS, andfunctionally effect or contribute to the nuclear translocation of theNLS-containing protein or complex can be used in the assays of theinvention. Fragments of such cellular proteins that bind NLS can also beused in the assays of the invention. Such cellular proteins andfragments thereof, collectively referred herein to as the cellularreceptor moiety, may include but are not limited to, karyopherin αβheterodimer and fragments thereof, or fusion proteins, such askaryopherin α-glutathione S-transferase (GST-karyopherin α).

[0145] The assays are performed in vitro in which the cellular receptormoiety is immobilized directly or indirectly onto a solid support. TheNLS-containing protein or molecular complex, purified or in a cellextract, is contacted with the immobilized cellular receptor moiety inthe presence of test compound. As a control, the NLS-containing proteinor molecular complex is contacted with the immobilized cellular receptormoiety under the same condition, but in the absence of the testcompound. After an interval sufficient for binding reactions to occuramong the components in the assay, the solid support is washed to removeany unbound molecules. A detection procedure is performed with the solidsupport to quantify the binding of NLS-containing protein or molecularcomplex to the immobilized cellular receptor moiety as compared tobinding reactions in the absence of test compound. The absence of boundNLS-containing protein or molecular complex, or a reduction in thebinding of the NLS-containing protein or molecular complex to the solidsupport, in the presence of a test compound, indicates that the testcompound can be useful in inhibiting the importation of the specificNLS-containing protein or molecular complex into the nucleus.

[0146] The detection procedure may employ an antibody or a ligand thatrecognizes and binds the NLS-containing protein or a component of themolecular complex. Alternatively, if the molecular complex comprisesnucleic acids, such as the case of HIV-1 pre-integration complex,polymerase chain reaction may be employed to detect the presence ofspecific nucleic acid sequences on the solid support. Any appropriateisotopic and nonisotopic labels can be used in conjunction with thedetection procedure. Detection or measurement of the antibody, ligand oramplified nucleic acids is accomplished by standard techniques wellknown in the art. Those skilled in the art will be able to determineoperative and optimal conditions for the above-described techniques byemploying routine experimentation.

[0147] For example, a screening assay for alkyl aryl carbonyl compoundsthat inactivate the NLS of HIV matrix antigen can be set up as follows.A glutathione S-transferase-karyopherin α fusion protein(GST-karyopherin α) that binds NLS is used as the cellular receptormoiety. The fusion-protein is immobilized onto glutathione Sepharose(Pharmacia) by incubation for 30-60 minutes at room temperature.Cytoplasmic extracts from cells that have been infected by HIV may beprepared by mechanically lysing infected cells in the presence ofprotease inhibitors, and removing the nuclei by centrifugation.

[0148] Alternatively, HIV MA and RT, and possibly other accessoryproteins, such as Vpr, that are present in the pre-integration complexcan be produced by recombinant DNA methods, and reconstituted in vitrointo a complex for use in the assay. The cytoplasmic extract or thereconstituted MA/RT complex is incubated at room temperature with theGST-karyopherin α Sepharose beads in the presence of variousconcentrations of compounds of the invention, for an interval sufficientfor binding interactions to occur, for example, 30 minutes. At the endof the incubation, the beads are washed to remove any unbound material.To quantitate the binding of pre-integration complex to the beads, HIV-1DNA was isolated from the beads by SDS-proteinase K treatment withsubsequent phenol-chloroform extraction, and analyzed by polymerasechain reaction using primers specific for the pol gene, as described inBukrinsky, M. I., et al., 1995, J. Exp. Med. 181:735-745. Alternatively,the amount of MA or RT bound to the beads can be determined by animmunoassay using anti-MA or anti-RT sera. Compounds of the inventionthat can be used to block nuclear importation of HIV are indicated by areduction or inhibition of binding to the beads by the HIV-1pre-integration complex or reconstituted MA/RT complex as compared tothe control.

6 EXAMPLES 6.1 SYNTHESIS OF SPECIFIC COMPOUNDS

[0149] Compound No. 2, FIG. 1A: A suspension of Compound No. 11(2-amino-4-(3,5-diacetylphenyl)amino-6-methylpyrimidine) (0.284 g), wassuspended in 1:1 acetonitrile-tetrahydrofuran was treated with methyliodide (2 mL) and heated at 40-45° C. under a reflux condenser for 18hr. Cooling and filtration gave 0.35 g of2-amino-4-(3,5-diacetylphenyl)amino-1,6-dimethylpyrimidinium iodide, mp292° C.

[0150]2-Amino-4-(3,5-diacetylphenyl)imino-1,4-dihydro-1,6-dimethylpyrimidine.A suspension of 21 g (49.3 mmole) of2-amino-4-(3,5-diacetylphenyl)amino-1,6-dimethylpyrimidinium iodide(compound No. 2, synthesized as described in section 6.1) in 1:1methanol/water (750 mL) at 60° C. was treated with excess 2N NaOH withcooling to maintain about 60° C. An additional 200 mL of water was addedand the mixture was cooled in ice and filtered to give 14.69 g2-amino-4-(3,5-diacetylphenyl)imino-1,4-dihydro-1,6-dimethylpyrimidineas yellow crystals, mp 219-220°.

[0151] 2-Amino-4-(3,5-diacetylphenyl)amino-1,6-dimethylpyrimidiniumchloride (CNI-0294). CNI-0294 is the chloride salt of compound No. 2.The base2-amino-4-(3,5-diacetylphenyl)imino-1,4-dihydro-1,6-dimethylpyrimidine(14.35 g, 48 mmole) was dissolved in 500 mL of methanol and treated withHCl gas until precipitation appeared complete. Filtration gave 12.8 g ofwhite crystals with a faint yellowish tinge, mp 306.5-307.50.

[0152] Compound No. 11 (CNI-1194): A suspension of 3,5-diacetylaniline(0.885 g) in water (18 mL) was treated with2-amino-4-chloro-6-methylpyrimidine (0.718 g) and concentrated HCl (0.42mL) and heated at 90-100° C. for 30 min. After cooling the mixture wastreated with 10 mL of aqueous 1N KOH. The mixture was stirred for 10 minand the solid was filtered out, washed with water, and dried, to give1.332 g of tan crystals. Recrystallization from ethylacetate-2-methoxyethanol gave 1.175 g of2-amino-4-(3,5-diacetyl-phenyl)amino-6-methylpyrimidine as light buffcrystals, mp 240-241° C.

[0153] Compound No. 12. A suspension of 3,5-diacetylaniline (0.531 g) inwater (8 mL) was treated with cyanoguanidine (0.285 g) and conc. HCl(0.25 mL) and heated at reflux. After 6 hr the mixture was cooled andconcentrated and 0.248 g of off-white solid was filtered out and driedto give N-(3,5-diacetylphenyl)biguanide hydrochloride, mp 260-70° C.(dec).

[0154] Compound No. 13: A suspension of 3,5-diacetylaniline (1.95 g) inwater (10 mL) was treated with 2-chloro-4,6-diamino-1,3,5-triazine(1.455 g) and concentrated HCl (0.1 mL) and heated at reflux for 20 min.After cooling the hydrochloride of Compound No. 13 separated as a whitepowder. This was filtered out, dissolved in 60 mL of boiling aqueous 75%methanol and treated with triethylamine (1.5 mL). On cooling, off-whiteflakes separated. Filtration and drying gave 1.79 g of2-(3,5-diacetylphenyl)amino-4,6-diamino-1,3,5-triazine, mp 271-2° C.

[0155] Compound No. 14 (CNI-H1494):4-(3-acetylphenyl)amino-2-amino-6-methylpyrimidine, Compound No. 15,(0.968 g) was suspended in acetone (5 mL) containing methyl iodide (2mL) was heated at reflux for 48 hr. Filtration after cooling gave 0.657g of 4-(3-acetylphenyl)amino-2-amino-1,6-dimethylpyrimidinium iodide asa white powder, mp 238-40° C.

[0156] Compound No. 15 (CNI-1594): A suspension of m-aminoacetophenone(2.7 g) and 2-amino-4-chloro-6-methyl-pyrimidine (2.87 g) in 40 mL waterwas treated with 1.7 mL concentrated HCl and heated at reflux for 1hour. Addition of 40 mL 1N KOH gave a light buff solid, which wasfiltered out and dried to give 3.8 g4-(3-acetylphenyl)amino-2-amino-6-methylpyrimidine, mp 196-98° C.

[0157] Compound No. 16: A suspension of 3,5-diacetylaniline (0.531 g) inwater (10 mL) was treated with 6-chloropurine (0.464 g) and concentratedHCl (0.25 mL) and heated at reflux for 30 min. After cooling the mixturewas treated with 6 mL of aqueous 1N KOH. The mixture was stirred for 10min and the solid was filtered out, washed with water, and dried, togive 0.80 g of 6-[(3,5-diacetylphenyl)amino]purine, mp dec 340-350° C.

[0158] Compound No. 17 (CNI-1794): A suspension of p-aminoacetophenone(1.35 g) and 2-amino-4-chloro-6-methyl-pyrimidine (1.435 g) in 20 mLwater was treated with 0.85 mL conc HCl and heated at reflux for 1 hr.Addition of 20 mL 1N KOH gave a light buff solid, which was filtered outand dried to give 2.28 g4-(3-acetylphenyl)amino-2-amino-6-methylpyrimidine, mp 194-196° C. Ofthis, 1.21 g was treated with methyl iodide (3 mL) in dimethylformamide(15 mL) at room temperature for 42 hr. Dilution with ethyl acetate andfiltration gave 1.11 g4-(4,acetylphenyl)amino-2-amino-1,6-dimethylpyrimidinium iodide as awhite powder, mp 302-3° C.

[0159] Compound No. 45. (CNI-4594) A mixture of aniline (0.93 g) and2-amino-4-chloro-6-methylpyrimidine (1.44 g) in 36 mL water was treatedwith 0.84 mL conc HCl and heated at reflux for 1 hr. Addition of 20 mL1N KOH gave a light buff solid, which was filtered out, dried, andrecrystallized from ethyl acetate/2-methoxyethanol and ethylacetate/hexane to give 0.69 g 4-phenylamino-2-amino-6-methylpyrimidine,mp 179-180° C.

[0160] Compound No. 46. A suspension of4-phenylamino-2-amino-6-methylpyrimidine, Compound No. 45, (0.25 g) inethanol (4 mL) was treated with methyl methanesulfonate (0.090 g) andheated at reflux for 5 days. Additional methyl methanesulfonate (0.090g) was added and the mixture refluxed another 2 days. Concentration andrecrystallization from a mixture of methanol, ethyl acetate, andtert-butyl ethyl ether gave 0.10 g of4-phenylamino-2-amino-1,6-dimethylpyrimidinium methanesulfonate.

[0161] 3,5-diacetylaniline (CNI-1894) was synthesized as per Ulrich etal. (1983, J Med Chem 27:35-40). Diacetylanilines substituted in otherpositions can be synthesized according to Ulrich et al. supra orMcKinnon et al. (1971, Can J Chem 49:2019-2022). All other startingmaterials were obtained from the Aldrich Chemical Co. Nuclear magneticresonance spectra and elemental analysis for all the compounds agreedwith expected values.

6.2 THE USE OF COMPOUND NO. 2 TO INHIBIT HIV REPLICATION IN PRIMARYMACROPHAGE LINES 6.2.1 Materials and Methods

[0162] Primary human monocytes were obtained from peripheral blood byFicoll-Hypaque centrifugation and adherence to plastic as describedpreviously. Gartner S. P., et al., 1986, Science 233:215. Briefly, afterFicoll-Hypaque (Pharmacia) separation, PBMCs were washed 4 times withDMEM (the last wash was done at 800 rpm to remove platelets) andresuspended in monocyte culture medium [DMEM supplemented with 1 mMglutamine, 10% heat-inactivated human serum, 1% penicillin+streptomycinmixture (Sigma)] at a density of 6×10⁶ cells/ml. Cells were seeded in24-well plates (1 ml per well) and incubated for 2 h at 37° C., 5% CO₂.Following incubation, cells were washed 3 times with DMEM to removenon-adherent cells and incubation was continued in monocyte culturemedium supplemented with 250 U/ml human M-CSF (Sigma). Cells wereallowed to mature for 7 days prior to infection with the monocyte-tropicstrain, HIV-1_(ADA). Nuovo, G. J., et al., 1992, Diagn. Mol. Pathol.1:98. Two hours after infection, cells were washed with medium andcultured in RPMI supplemented with 10% human serum. In experiments wherePCR analysis was performed, virus was pretreated with RNAse-free DNAse(Boehinger-Mannheim) for 2 h at room temperature and then filteredthough a 0.2 μm pore nitrocellulose filter prior to infection.

[0163] PBMCs were purified by Ficoll-Hypaque centrifugation andactivated by 10 μg/ml PHA-P (Sigma) and 20 U/ml recombinant human IL-2(rhIL-2) in RPMI 1640 supplemented with 10% FBS (HyClone). After 24 hincubation, cells were washed and inoculated with HIV-1_(ADA) in RPMI1640 supplemented with 10% FBS. After a 2 h adsorption, free virus waswashed away and cells were cultured in RPMI 1640 supplemented with 10%FBS and 20 U/ml rhIL-2.

[0164] Virus stock and infection. Macrophage-tropic strain HIV-1_(ADA)was amplified in primary human monocytes and concentrated to producestock with TCID₅₀ of about 10⁵/ml. The concentration of HIV-1 wasdetermined by immunoassay of viral p24, concentration; using aconversion factor of 1 ng/200 HIV-1 particles.

6.2.2 p24 and RT Assay

[0165] For p24 assay, sequential 1:9 dilutions of culture supernatantwere prepared and analyzed by ELISA as suggested by the manufacturer(Cellular Products, Buffalo, N.Y.). For the reverse transcriptase (RT)assay, 10 μl of culture supernatant was added to 40 μl of reactionmixture (final composition was 50 mM Tris-HCl, pH 7.8; 20 mM KCl; 5 mMMgCl₂; 1 mM DTT; 0.1% Triton X-100; 0.2 OD/ml polyA; 0.2 OD/mloligo(dT)₁₂₋₁₈; and 40 μCi/ml ³H-dTTP (76 Ci/mmol, DuPont) and incubated2 hr at 37° C. 5 μl of the reaction mixture was then spotted onto the DE81 (Whatman) paper. Paper was air dried and washed 5 times with 5%Na₂HPO₄₁ followed by rinsing with distilled water. After air drying,paper was put on a Flexi Filter plate (Packard), covered withscintillation fluid and counted in a Top Count Microplate Counter(Packard). Results are expressed as counts per minute in 1 ml ofsupernatant (cpm/ml).

6.2.3 Results Dividing and Quiescent Cells

[0166] The cytotoxicity of Compound No. 2 was tested in monocytecultures by trypan blue exclusion assay or lactate dehydrogenase (LDH)release. By both assays, no cytotoxic effect was observed withconcentrations of the compound up to 10 μM (data not shown). Resultspresented in FIG. 2 show the effect of various concentrations ofCompound 2 on HIV-1 replication in monocytes. From this experiment, weestimate the IC₅₀ for this compound between 0.1 and 1 nM. Similar andhigher concentrations of the compound were also tested on activatedPBLs. The anti-viral effect of this compound was much less expressed inthese actively dividing cell populations (FIG. 3) No anti-viral effectwas detected when cultures of replicating cells were infected at themultiplicity of infection used to infect monocytes.

6.2.4 AZT and Compound No. 2 in Combination

[0167] AZT is a drug that is routinely used to treat HIV-1 infectedpersons. However, two factors are known to diminish the effectiveness ofAZT: its toxicity and the emergence of resistant mutant strains ofHIV-1. The effects of both of these factors can be reduced byadministering a second, synergistic HIV-1-inhibitory drug with AZT.

[0168] In view of these premises, the effects on HIV-1 replication inhuman monocyte cultures of the various concentrations of AZT, alone orin combination with 100 nM Compound No. 2, were tested using theprotocols of Sections 6.2.1 and 6.2.2. Drugs were added to the monocytecultures together with HIV-1 at about 10⁵ TCID/ml. The concentration ofdrugs was maintained on refeeding. HIV-1 replication was assessed byassay of the supernatant for reverse transcriptase activity. The resultsare expressed as mean±std. dev. (cpmx10⁻³) in Table V. TABLE V Effectsof Combined AZT/Compound No. 2 on HIV-1 infected Monocyte Cultures day-7day-11 [AZT] (−) No. 2 (+) No. 2 (−) No. 2 (+) No. 2 0 1.46 ± 0.43 0.37± 0.07 1.81 ± 0.75 0.72 ± 0.30 10 pM 0.92 ± 0.21 0.15 ± 0.05 1.63 ± 0.810.18 ± 0.06 100 pM 0.79 ± 0.14 0.13 ± 0.04 1.34 ± 0.59 0.15 ± 0.06 1 nM0.60 ± 0.28 0.04 ± 0.02 1.07 ± 0.49 0.09 ± 0.03 10 nM 0.05 ± 0.02 0.03 ±0.02 0.08 ± 0.03 0.07 ± 0.03

[0169] These results demonstrate that there is synergy between the AZTand Compound No. 2. The synergistic effects are most pronounced at thelower doses of AZT on day 11. For example, 10 pM AZT alone produces anabout 20% reduction in RT activity on day-11, 100 nM Compound No. 2alone produces about a 60 k reduction. Without synergy, the combinationshould produce a 70% reduction (100×(1−(0.8×0.4)). Instead the observedreduction was 90 k.

6.3 THE COMPOUNDS OF THE INVENTION DO NOT BLOCK THE NUCLEAR IMPORTATIONOF ESSENTIAL PROTEINS IN CELLS 6.3.1 Direct Demonstration of theInhibition of HIV-1 Nuclear Importation by Compound No. 2

[0170] The effects of Compound No. 2 on the nuclear importation of HIV-1preintegration complexes can be directly measured by detecting thepresence of circularized duplex HIV-1 genomic DNA. These duplex circlescan be readily detected by PCR amplification using primers which spanthe junction of the 1 circularized HIV-1 genome. Bukrinsky, M. I., etal., 1992, Proc. Natl. Acad. Sci. 89:6580-84.

[0171] Briefly, the efficiency of nuclear translocation was estimated bythe ratio between the 2-LTR and pol-specific PCR products, whichreflects the portion of 2-LTR circle DNA molecules as a fraction of theentire pool of intracellular HIV-1 DNA. Viral 2-LTR circle DNA is formedexclusively within the nucleus of infected cells and thus is aconvenient marker of successful nuclear translocation. Bukrinsky, M. I.,1992, Procd. Natl. Acad. Sci. 89:6580-84; Bukrinsky, M. I., 1993, Nature365:666-669.

[0172] PCR analysis of HIV-1 DNA: Total DNA was extracted fromHIV-1-infected cells using the IsoQuick extraction kit (MicroprobeCorp., Garden Grove, Calif.). DNA was then analyzed by PCR using primerpairs that amplify the following sequences: a fragment of HIV-1(LTR/gag) that is the last one to be synthesized during reversetranscription and therefore represents the pool of full-length viral DNAmolecules; a fragment of polymerase gene (pol); a 2-LTR junction regionfound only in HIV-1 2-LTR circle DNA molecules; or a fragment of thecellular a-tubulin gene. Dilutions of 8E5 cells (containing 1 integratedcopy of HIV-1 DNA per genome) into CEM cells were used as standards.Amplification products were transferred to nylon membrane filters andhybridized to ³²P-labeled oligonucleotides corresponding to internalsequences specific for each PCR amplification fragment, followed byexposure to Kodak XAR-5 film or a phosphor screen.

[0173] Quantitation of PCR Reactions: Bands of correct size revealedafter hybridization were quantitated with a PhosphorImager (MolecularDynamics) by measuring the total density (integrated volume) ofrectangles enclosing the corresponding product band. Efficiency ofnuclear translocation of HIV-1 DNA was estimated by measurement of theamount of 2-LTR circle DNA (N_(2-LTR)) relative to total viral DNA(N_(tot)) in each culture, indexed to the same ratio of appropriatecontrol cultures. Thus, Translocation Index=(N_(2-LTR)/N_(tot))(C_(2-LTR)/C_(tot))×100.

[0174] Results: Primary human monocytes were infected with HIV-1_(ADA)in the presence of 100 nM concentration of Compound No. 2 or withoutdrugs (control). Half the medium was changed every 3 days, and drugswere present throughout the whole experiment. Cell samples were taken at48 and 96 hours post infection and the Translocation Index, relative tothe drug free control was determined. At both time points theTranslocation Index was less than 10, indicating there was greater than90% inhibition of nuclear importation.

7 PHARMACOKINETIC AND TOXICOLOGICAL STUDIES

[0175] This section describes in detail the techniques that were used tostudy the toxicological and pharmacological properties of the compoundsof the invention.

7.1 Drug Analysis

[0176] Standard addition curves for each test compound were constructedby adding increased amounts of drug to mouse or human A⁺ plasma (LongIsland Blood Services; Melville, N.Y.). An equal volume of 10 mMtetramethylammonium chloride/10 mM heptane sulfonate/4.2 mM H₃PO₄(Buffer A) was added to the plasma sample, which was then loaded onto awashed 1 g cyanopropylsilane (or octadecylsilane for CNI-1894)solid-phase extraction column (Fisher Scientific). The column was washedwith 1.0 ml of water and then eluted with 1.0 ml of 10 mMtetramethylammonium chloride/10 mM heptane sulfonate/4.2 mM H₃PO₄/95%CH₃CN/5% H₂O (Buffer C). The eluted sample was reduced to dryness in arotary evaporator and resuspended in 1.0 ml Buffer A.

[0177] Two hundred μl of the resuspended sample was injected onto aHewlett-Packard model 1090 high performance liquid chromatography system(HPLC)(Wilmington, Del.) equipped with a photodiode arrayultraviolet/visible spectrophotometric detector, autosampler, andChemstation operating software. The column used was a 250×4.6 mm ZorbaxRX-C8 column (Mac-Mod Analyticals.; Chadd's Ford, Pa.) kept at roomtemperature and run at 1.5 ml/min. The mobile phase used was Buffer Aand 10 mM tetramethylammonium chloride/10 mM heptane sulfonate/4.2 mMH₃PO₄/75% CH₃CN/25% H₂O (Buffer B), with all runs initiated at 10%Buffer B. A linear 30 min gradient to 60% Buffer B was then performed,followed by a 4 min reverse gradient to initial conditions. CompoundsCNI-0294, -1194, 1594, and -1794 were detected by ultraviolet absorbanceat 300 nm, CNI-1894at 240 nm, and pentamidine at 265 nm. In this assaysystem, the CNI test compounds have a linear response and are detectabledown to at least 19.5 ng per injection.

7.2 TOXICITY STUDIES 7.2.1 Method

[0178] The doses of compounds of the invention found to be lethal to 50%of the mice (LD₅₀) were determined by intraperitoneal injection ofgroups of five animals with increasing doses of each compound. CNI-0294was administered from 0, 2, 10, 20, 40, 80, 160, 320, 640, 1280 mg/kg in0.5 ml of water/HCl; CNI-1594 at 0, 2.4, 5, 10, 20, 40, 80 mg/kg in 0.5ml of water/HCl; CNI-1794 at 0, 20, 50, 80 mg/kg in 0.5 ml of water/HCl;and CNI-1894 at 0, 10, 20, 40, 80, 240, 480, 960 mg/kg in water/HCl. Allanimals were observed for visible signs of acute or long-term toxicity.The percentage of animals in each group which died were utilized tocalculate the LD₅₀ by non-linear curve fitting with the Enzfit software(Elsevier Bioscience; Cambridge, UK) programmed with the Chou equation(Chou 1976, J Theor Biol 39:253-276)).

7.2.2 Results

[0179] The compounds (FIGS. 4A-E), were screened for toxicity via amodified LD₅₀ assay procedure as described above in an outbred strain ofmice. The results are shown in Table VII as follows: TABLE VI Thetoxicity of the CNI compounds, as measured by the median lethal dosedetermined as described above. LD₅₀ ± standard deviation Compound(mg/kg) 0294 587.77 ± 65.79 1194 >160* 1594 49.04 ± 0.08 1794 48.93 ±0.12 1894 258.64 ± 1.37 

[0180] CNI-0294 was found to be very well tolerated (see Table VI), withno overt signs of toxicity detectable at doses approaching the LD₅₀. Theother compounds in the CNI series were designed to allow forstructure-function relationships with respect to activity and toxicity.CNI-1194, which differs from CNI-0294 only by the lack of a methyl groupon the heterocyclic nitrogen, was also well tolerated, with a high LD₅₀(Table VI). However, CNI-1594, which is similar to CNI-1194 plus theomission of one of the acetyl groups on the benzene rings, wasappreciably more lethal (Table VI). This toxicity was immediate, withdeath occurring in minutes and the animals displaying signs of acuteneurotoxicity. CNI-1794, which is identical to CNI-1594 except that thesingle acetyl group is moved para to the heterocyclic substituent, hadan LD₅₀ identical to that for CNI-1594 (Table VI). CNI-1894, which issimilar to CNI-0294 and -1194 but lacks the heterocyclic ring, was alsoreasonably well tolerated. Animals dosed with large amounts of CNI-1894died 2-3 days post injection, and showed no sign of any immediatetoxicity. Based on the above observation, it is concluded that thepresence of the heterocyclic ring in the compounds of the inventionplays only a small role in determining toxicity, while the presence oftwo acetyl groups on the benzene ring is very important. Therefore, apreferred compound of the invention showing low toxicity contains twoacetyl groups on the benzene ring.

7.3 PHARMACOKINETIC STUDIES 7.3.1 Methods

[0181] Female ND4 Swiss Webster mice (21-24 g) were obtained from HarlanSprague Dawley (Indianapolis, Ind.) and randomly placed in groups offive in cages with free access to food and water. Each group of animalsreceived 50 mg/kg of CNI-0294, -1194, or -1894, or 20 mg/kg of CNI-1594in a volume of 0.5 ml. Compound CNI-0294 was administeredintraperitoneally or by oral gavage as a solution in water or asuspension in 10% DMSO/peanut oil. The other CNI compounds wereadministered intraperitoneally or by oral gavage as a solution in watertitrated with sufficient HCl to dissolve the drug. At various timepoints, ranging from 5 min to 4 days, a single group of animals waseuthanized by carbon dioxide inhalation and bled by cardiac punctureusing heparin as an anticoagulant. The blood from the five mice in thegroup was pooled and centrifuged at 14000×g for 10 min. The volume ofplasma was measured, and equal volume of Buffer A added, and the mixtureextracted and analyzed as described above, except that the dried eluateswere resuspended in 200 μl Buffer A and 100 μl was injected onto thehigh performance liquid chromatography (HPLC) system.

[0182] As inspection of the blood concentration-time curves for a singlei.p. injection showed a typical biphasic appearance, standard methods ofpharmacokinetic measurement were employed (1982, Gibaldi et al.,Pharmacokinetics. Marcel Dekker, New York). The area under the plasmaconcentration-time curve (AUC) was determined, and bioavailability wasmeasured as AUC_(oral)/AUC_(i.p.) A and B represent the zero timeintercept of the distribution and elimination phases respectively, and aand β the respective slopes of the phases multiplied by 2.303. Thet_(1/2≢α) and t_(1/2β) are calculated half-lives of the drug in eachphase (0.693/α and 0.693/β respectively). The volume of distribution(VD) was calculated as dose/B, and the total clearance rate (Cl_(tot))calculated as β*V_(D). C_(max) and t_(max) are the maximal plasmaconcentration and time of this measurement, respectively.

7.3.2 Results

[0183] As judged by the plasma concentration-time curves from a singleintraperitoneal injection, each compound in the CNI series had similarpharmacokinetic properties despite the structural differences. Thekinetic parameters are summarized in Table III and a typical pattern isshown for CNI-1194 in FIG. 5. The drugs were rapidly absorbed, with themaximal plasma concentration reached in 5-15 min, and also had a rapiddistribution phase, with a t_(1/2α) of 0.32-0.62 hr. Differences werefound to occur in the maximal plasma concentration and parametersrelated to the elimination phase. CNI-0294 achieved the highest maximalplasma level for a single 50 mg/kg i.p. injection, with 18.76 μg/ml, andCNI-1894 was very similar with a value of 13.43 μg/ml. As CNI-1194 hadan appreciably lower maximal plasma level and a slower t_(max) whencompared with CNI-0294, it appears that the presence of the methylsubstituent on the heterocyclic nitrogen enhances drug absorption fromthe peritoneum. A comparison of CNI-1194 and CNI-1594 implied that thenumber of acetyl groups had little effect on drug absorption. The valuesrelating to elimination (β, B, t_(1/2β), V_(d), Cl_(tot)) were found tovary, but no clear structural relationship could be discerned. All thecompounds, except CNI-1894, were undetectable in plasma after 24 hr andapproached the limit of detection after 5-6 hr. Therefore, as a generalproperty, the compounds of the invention are absorbed and eliminatedrapidly. A preferred compound of the invention has a methyl substituenton the heterocyclic ring nitrogen at position 1 and possesses enhancedabsorption from the peritoneum.

[0184] Experiments were also performed with CNI-0294 and -1194 toevaluate relative bioavailability. By comparing the AUC_(oral) againstthe AUC_(i.p.) for a single 50 mg/kg dose, CNI-0294 was found to have 6%relative bioavailability and CNI-1194 15%. The maximal plasma level was0.4 μg/ml for CNI-0294 and 0.35 μg/ml for CNI-1194, and the drugs weredetectable in plasma for at least 6 hr (see FIG. 5).

7.4 METABOLIC STUDIES

[0185] During the analysis of the plasma samples for the pharmacokineticparameters, a number of additional HPLC peaks were detected whichincreased and decreased over time. Extra peaks of this nature were seenin samples from each of the CNI series as shown in FIGS. 8A-8D. As itwas possible that these peaks represented metabolites of the CNIcompounds, the compounds of the invention were screened in a simplemodel of primary metabolism.

7.4.1 Method

[0186] Several female ND4 Swiss Webster mice were euthanized by carbondioxide inhalation and the livers excised and rinsed with ice coldphosphate buffered saline (pH 7.4). The livers were minced, gentlyhomogenized in 50 mM phosphate buffer (pH 7.4) with a Douncehomogenizer, and centrifuged at 9600×g for 20 min. Thepost-mitochondrial supernatant was kept, glycerol added to 20%, andfrozen at −70° C. in 1.0 ml aliquots until used. For each incubation,1.0 ml of a 1.0 mg/ml drug solution was added to 3.0 ml of 50 mMphosphate buffer (pH 7.4), 1.0 ml of 2 mg/ml NADPH in 50 mM phosphate(pH 7.4), and 1.0 ml of the post-mitochondrial supernatant. Five hundredμl of each incubate was then immediately transferred to an ice-cold tubeto provide the zero-time sample, and addition 500 μl aliquots removed toice-cold tubes at 8, 15, 30, and 60 min. The samples were thenextracted, and analyzed by HPLC as described in section 7.1. Controlincubations were also performed where drug or post-mitochondrialsupernatant was omitted. An incubation using pentamidine was performedto confirm microsomal activity (Berger et al., 1992, Antimicrob. Ag.Chemother. 36:1825-1831). Peaks in the CNI compound incubations whichincreased over time, and were not present in control samples lacking theenzyme preparation were treated as putative metabolites.

7.4.2 Results

[0187] Using post-mitochondrial supernatants of homogenized mouse liversas a source of enzyme, the drugs were incubated in the presence ofNADPH. As described in Berger et al. supra, pentamidine was used as apositive control, and the seven, expected, primary metabolites weredetectable, confirming the activity of the enzyme preparation.Extraction and analysis of the CNI incubates showed the presence ofnumerous, putative metabolite peaks that were not present in negativecontrol incubations (FIG. 6). Incubation of CNI-0294, -1594, or -1194was found to produce three minor and one major metabolite and CNI-1894had one minor and one major metabolite. The major metabolite was foundto elute 0.9-1.2 min closer to the solvent front for CNI-0294, -1194,and -1594, suggesting that the same position was being altered in eachof these compounds. The metabolic conversion in the post-mitochondrialsupernatant system was considerable, with 43.5% of CNI-0294, 65.19% ofCNI-1194, 11.74% of CNI-1594, and 17.28% of CNI-1894 altered during thecourse of a 60 min incubation (as judged by peak area). These resultsindicated that appreciable metabolism of the compounds of the inventionshould occur in vivo.

[0188] Re-examination of the plasma samples confirmed that the severalof the unknown plasma peaks seen in FIGS. 6A and 6B corresponded to theputative metabolites in FIGS. 7A-7D. However, the metabolic model systemdid not produce all the unknown peaks seen in the plasma samples. Inparticular, a plasma peak eluting at 11-14 min was seen with all thecompounds in vivo, but not seen at all in the in vitro test system. Aswas evident from the plasma time-course samples, there appeared to be alarge amount of metabolic conversion in vivo of all of the compounds,regardless of the route of administration.

7.5 CONCLUSIONS

[0189] The toxicity, pharmacokinetics, and metabolism of the novelarylene bis(methylketone) compounds of the invention, and several novelanalogues thereof likewise of the invention were examined in mice. Witha median lethal dose of 587.77 mg/kg, CNI-0294 was well tolerated whenadministered intraperitoneally. Analogues which also had two acetylgroups on the phenyl moiety were also well tolerated, with median lethaldoses exceeding 160 mg/kg i.p. All visible toxic reactions appeared tobe rather delayed (generally 2-3 days post injection). While no biopsysamples were taken, such a delay would be consistent with organ damageby very high doses these compounds. Compounds which had only one acetylgroup were found to be more toxic, with median lethal doses of48.93-49.04 mg/kg i.p. While the visible symptoms following injection ofCNI-1594 or -1794 suggested a lethal neurotoxicity, the structuraldifferences between the two drugs indicate that antagonism of anendogenous neurotransmitter is unlikely.

[0190] In test animals, all of the compounds possessed very rapidpharmacokinetic properties, with the plasma maximal concentration, forintraperitoneal injection, being reached in 5-15 min, and 15-60 min fororal dosing. For CNI-0294, a plasma maximal concentration of 18.76-18.93μg/ml was reached after injection of 50 mg/kg i.p. The other compoundstested achieved lower maximal plasma levels (1.9-13.43 μg/ml). Thehalf-life of the distribution phase (t_(1/2α)) was 0.32-0.62 hours, andthat for the elimination phase (t_(1/2β)) was 3.65-23.10 hours. All ofthe kinetic parameters are consistent with drugs that are very rapidlycleared from the plasma and are not retained in tissues for a longperiod of time. Both CNI-0294 and -1194 were orally absorbed, with arelative bioavailability of 6 and 15 percent respectively. This latterfeature is very favorable for continued development of these compoundsas anti-infective agents, particularly as antiviral and antiparasiticagents, and more particularly as anti-retroviral and anti-protozoalagents, and yet particularly as anti-HIV agents and antimalarials. Thetoxicity, kinetic, and bioavailability data suggest that frequent, high,oral doses of the CNI-0294 can safely maintain therapeutically effectiveplasma concentration.

[0191] Metabolism of the drugs was assessed in a mouse liverpost-mitochondrial supernatant system, and extensive metabolism wasdiscovered (11.74-65.19% metabolized during, a 60 minute incubation).Examination of plasma samples showed that there was considerable in vivometabolism, with at least 4-6 metabolites easily detected during thefirst 3 hours following i.p. administration of the test compounds. Thelevels of metabolite rapidly exceeded plasma concentrations of theparent compound. The HPLC retention times indicated that the compoundswere likely altered in the same positions. In addition, the metabolites,like the parent compounds, appeared to have very rapid plasma kinetics.

8 EXAMPLE: DEMONSTRATION OF ANTI-MALARIAL ACTIVITY 8.1 THE COMPOUNDSHAVE ANTI-MALARIAL ACTIVITY IN VITRO 8.1.1 Method

[0192] The antimalarial activity of the compounds was determinedessentially as described in Desjardins et al. supra. Fifty μl of variousconcentrations of a compound of the invention, chloroquine, orpyrimethamine were added to the wells of microtiter plates, followed by200 μl of ring-stage, synchronized, P. falciparum-infected erythrocytes(final hematocrit=1.5%, final parasitemia =1-5%). The plates wereincubated for 24 hr in a candle jar kept at 37° C., and then 25 μl of[³H]-hypoxanthine (Amersham, Arlington Heights, Ill.; 2.5 μl Ci/well)was added. The plates were then incubated for a further 24 hr, beforeharvesting onto Unifilter-96 GF/C filter-microplates (Packard; Meriden,Conn.). Twenty-five μl of Microscint scintillation fluid (Packard) wasadded to each well of the filter-microplate, which was subsequentlycounted in a Top-count microplate scintillation counter (Packard). Thepercent of [³H]-hypoxanthine uptake relative to controlinfect-erythrocytes was used to determine the IC₅₀ value for thecompounds by non-linear regression for LD₅₀ determination.

8.1.2 Results

[0193] Using the hypoxanthine-incorporation method for assessingPlasmodium falciparum growth in vitro as described above, CNI-0294 wasfound to have considerable anti-malarial activity (Table VII). TABLE VIIThe antimalarial activity of CNI- 0294, chloroquine, and pyrimethaminein vitro against several Plasmodium falciparum clones. The medianinhibitory concentration was determined as described above.Pyrimethamine CNI-0294 Clone Chloroquine IC₅₀ IC₅₀ (μM) IC₅₀ (μM) D1026.99 ± 2.42* 170.70 ± 24.60  4.00 + 0.41 Dd2 122.54 ± 7.26  103.70 ±9.79  3.52 + 0.10 FCR-3 104.68 ± 9.98  0.04 ± 0.01 3.09 + 0.30 HB3 6.73± 0.16 8.97 ± 2.75 1.79 + 0.27 W2mef 143.79 ± 13.30  17.81 ± 13.462.29 + 0.22

[0194] The median inhibitory concentration (IC₅₀) for CNI-0294 wascalculated to be 1.79-4.00 μM for a series of cloned parasites whichhave different sensitivities to chloroquine or pyrimethamine (TableVII).

[0195] The Dd2 clone of P. falciparum, which was both chloroquine andpyrimethamine resistant, was utilized to compare the antimalarialactivity of the remaining CNI compounds (Table VIII). TABLE VIII Theantimalarial activities of the CNI compounds against the chloroquine-and pyrimethamine- resistant P. falciparum clone Dd2. The medianinhibitory concentration was determined as described above. CompoundIC₅₀ ± standard deviation (μM) 0294  3.67 ± 0.57* 1194 20.27 ± 1.62 159423.73 ± 0.59 1894 ≧200** 4594 25.11 ± 0.72

[0196] In independent measurements, CNI-0294 agreed well with theresults in Table VII, and CNI-1194 was found to be approximately 5-foldless active. This difference suggested that the heterocyclic methylgroup is required for maximal activity. CNI-1594 had an IC₅₀ equal tothat for CNI-1194 or CNI-4594 demonstrating that loss of one or both ofthe acetyl groups can have little effect on the antimalarial activity.CNI-1894, however, was inactive at the highest concentration tested.

8.2 THE COMPOUNDS HAVE ANTI-MALARIAL ACTIVITY IN VIVO 8.2.1 Method

[0197] The antimalarial activity of CNI-0294 in vivo was assessed byinfecting female ND4 Swiss Webster mice with 100 μl of Plasmodiumberghei NYU-2 infected mouse erythrocytes (50% parasitemia) byintraperitoneal injection. The animals were subsequently injectedintraperitoneally once per day on days 1-4 of the infection with 0.5 mlwater or 0.5 ml of 50 mg/kg CNI-0294 in water. Four hours after thefinal injection, small blood samples were taken from the tail, and thinsmears stained with Dif-Quick (Baxter, Miami, Fla.). The parasitemia ofcontrol and treated animals was enumerated by inspection of at least1000 erythrocytes in each animal.

8.2.2 Results

[0198] As the CNI-0294 IC₅₀ for P. falciparum was in the range achievedfor approximately one hr following a single i.p. injection of 50 mg/kgin mice, the compound was also screened in vivo in mice infected withPlasmodium berghei. Utilizing the four day suppression test, whereparasitemia is enumerated following four daily injections of the testcompound (in this case 50 mg/kg i.p.), CNI-0294 was found tosignificantly (P≦0.01) lower the parasitemia by 10-fold (FIG. 9).

8.3 CONCLUSIONS

[0199] As indicated in Table VII, CNI-0294 was effective against variousclones of P. falciparum. The consistency in CNI-0294 IC₅₀ over such arange of chloroquine and pyrimethamine IC₅₀'s suggested that CNI-0294had a different mechanism of action than either of these establishedantimalarials.

[0200] While daily 50 mg/kg injections i.p., for 4 days, were found tostrongly suppress P. berghei infection in mice, these animals were notcompletely cured during this course of treatment. The difference betweenthese in vivo results and the more striking P. falciparum in vitroresults are likely due to the kinetic and metabolic properties of thecompound. In vitro, the parasites are exposed to a constant level of thedrug for 48 hr, with no source of host metabolizing enzymes. In the casein vivo, the single, daily i.p. injection only provides therapeuticplasma concentrations for approximately one hour and there isconsiderable metabolism to compounds which may have reducedanti-plasmodial activity. In light of these observations, one ofordinary skill in the art would be able to further optimize the dosingregimens.

9 EXAMPLE: MECHANISM OF INHIBITION OF HIV-1 NUCLEAR TRANSLOCATION BYCOMPOUND NO. 2

[0201] The following experiments demonstrate the inhibitory mechanism ofcompound No. 2 (also known as CNI-0294 or CNI-H0294) on HIV nuclearlocalization, which is based on the inactivation of the nuclearlocalization sequence (NLS) of the HIV matrix antigen (MA) in thepresence of HIV reverse transcriptase (RT). The results described hereinprovide a basis for the development of a novel class of antiviralcompound that inhibit nuclear localization and that are selectiveagainst specific NLS-containing proteins or molecular complexescomprising NLS-containing protein.

9.1 MATERIALS AND METHODS

[0202] Infection with Mutant HIV-1 or HIV-like Pseudovirions

[0203] H9 cells were infected with HIV-1 or HIV-like pseudovirions(Haffar, et al., 1990, J. Virol., 64:2653-2659) at a multiplicityadjusted according to p24 content (50 ng p24 per 106 cells). The MA NLS⁻virus contains substitutions of isoleucine residues for lysines inpositions 26 and 27 of MA in an NLHX backbone (Westervelt., et al.,1992, J. Virol. 66:2577-2582), thus inactivating the NLS. The Vpr⁻ virushas the initiating ATG of the vpr gene changed to GTG, thus abolishingexpression of this gene. The ΔMA NLS pseudovirions have leucinesubstituting for lysine in position 28 of MA. This mutation abrogatesnuclear translocation of HIV-1 gag RNA in growth-arrested H9 cellsinfected with pseudovirions. After a 1 hour absorption, excess virusesor pseudovirions were washed away, and cells were incubated for anadditional 2-3 hour period at 37° C. prior to analysis.

[0204] Preparation of Cytoplasmic Lysates

[0205] Cytoplasmic extracts were prepared by lysing cells in coldextraction buffer (10 mM KCl, 10 mM Tris-HCl [pH 7.6], 0.5 MM MgCl₂₁ 1μg/ml each of leupeptin and aprotinin, and 1 mM phenylmethylsulfonylfluoride [PMSF]) by 20-30 strokes of a Dounce homogenizer under thecontrol of phase-contrast microscopy. After removal of nuclei,cytoplasmic extract was cleared by centrifugation at 15,000 g for 10min.

[0206] Analysis of Binding of Nucleoprotein Complexes to Karyopherin α

[0207] Cytoplasmic extracts prepared from HIV-1-or pseudovirion-infectedH9 cells were adjusted to 0.14 M NaCl, 0.1% Tween 20 and precleared withglutathione-Sepharose beads for 30 min. at room temperature. Karyopherina was expressed as a fusion protein with glutathione S-transferase (GST)which can bind glutathione beads in solution. GST-karyopherin αimmobilized on Sepharose beads was then added (about 50 μg ofimmobilized karyopherin α per extract from 108 infected cells) and themixture was incubated at room temperature for another 30 min. Beads werethen pelleted by centrifugation and washed 3 times with PBS supplementedwith 0.1 k Tween 20, 1 μg/ml each of leupeptin and aprotinin, and 1 mMPMSF. HIV-1 DNA was isolated from the beads by SDS-proteinase Ktreatment with subsequent phenol-chloroform extraction, whilepseudovirion gag RNA was isolated by RNazol (Biotecx Laboratories Inc.).

[0208] Analysis of CNI-H0294 Interaction with HIV-1 Proteins in Solution

[0209] 0.28 nanomoles of recombinant MA or RT [p66/p51 dimer were mixedwith 20 nmol of [¹⁴C]CNI-H0294 (specific activity 5×10⁴ cpm/nmol) andincubated 2 hr at room temperature in 40 μl of binding buffer (PBSsupplemented with 1% BSA, 0.1% Tween 20, 1 μg/ml leupeptin, 1 μg/mlaprotinin, 1 mM PMSF). Sheep anti-MA or rabbit anti-RT sera (bothobtained from NIH AIDS Research and Reference Reagent Program) orpre-immune control sera were then added (at 1:100 dilution) andincubation continued for another 1 hr at room temperature.

[0210] Immune complexes were precipitated with protein G-agarose,washed, and then eluted with 0.1 M glycine, pH 2.8. Radioactivity of theeluate was measured in a scintillation counter.

[0211] Analysis of CNI-H0294 Interaction with HIV-1 Pre-integrationComplexes

[0212] Cytoplasmic lysates prepared from HIV-1-infected cells weretreated with 10 μM of [¹⁴C]-labeled CNI-H0294 (specific activity 5×10⁴cpm/nmol) in 1 ml extraction buffer adjusted to 0.14 M NaCl. Sodiumborohydride was then added to a final concentration 10 mM and sampleswere incubated 1 hr at room temperature prior to immunoprecipitation toreduce double bonds of Schiff bases to an irreversible secondary amine.Immunoprecipitation was performed as described above, but beads werestringently washed three times with PBS supplemented with 0.1% SDS, 1%sodium deoxycholate, 1 μg/ml each of aprotinin and leupeptin, and 1 mMPMSF.

9.2 RESULTS

[0213] CNI-H0294 reacts with adjacent lysines in the NLS, thus making itcapable of neutralizing NLSs on many different proteins. Interestingly,CNI-H0294 Exhibited remarkably low cytotoxicity in monocyte and Tlymphocyte cultures in vitro (50% toxic dose >1 mM) and in vivo in mice(LD50=590 mg/kg, see Table VI). These results suggest that the molecularmechanism of MA NLS inactivation by CNI-H0294 is very specific. Indeed,this compound, did not block nuclear import of nucleoplasmin-coated goldparticles, nor of BSA with conjugated NLS peptides that mimic the NLS ofSV40 large T antigen.

[0214] CNI-H0294 Inhibits Interaction Between HIV-1 Pre-integrationComplexes and Karyopherin α but does not Affect Binding of Karyopherin αto Pseudovirion-derived Nucleoprotein Complexes.

[0215] The initial step in the process of nuclear import is binding ofkaryopherin α (also termed NLS-receptor/importin) to an NLS. Resultspresented in FIG. 10A demonstrate that wild-type HIV-1 pre-integrationcomplexes bound to GST-karyopherin α immobilized onglutathione-Sepharose beads (lane 1). Mutant pre-integration complexesthat lack Vpr (MA NLS⁺Vpr⁻, lane 3) bound with reduced efficiency, whilebinding of the complexes with mutated MA NLS (MA NLS⁻Vpr⁺, lane 2) waseven more impaired. Pre-integration complexes that lack Vpr and aremutant in MA NLS (MA NLS⁻Vpr⁻) did not bind to karyopherin α (lane 4).These results are consistent with the analysis of MA and Vpr binding tokaryopherin α which demonstrated that while Vpr can bind weakly tokaryopherin α, its main role is to enhance the MA NLS-karyopherin αinteraction.

[0216] To facilitate analysis of HIV-1 nuclear translocation and of themechanism of drug effects on this process, a simplified model of theHIV-1 pre-integration complex was used which comprises a minimal numberof non-essential proteins. This model employs gag-env pseudovirionswhich exhibit an HIV-like core but are composed exclusively of Gag (MA,CA, NC, p6) and Env (gp41 and gp120) proteins. These pseudovirionspackage HIV-1 gag RNA and translocate this RNA into the nucleus of aninfected cell in a manner similar to the behavior of HIV-1pre-integration complexes. Results presented in FIG. 10B demonstratethat karyopherin α binds nucleoprotein complexes formed inpseudovirion-infected CD4+ T cells (lane 3). Binding required afunctional MA NLS as mutation of the NLS (FIG. 10A, lane 1) orpre-treatment of nucleoprotein complexes with polyclonal anti-MAantibodies (lane 2) greatly diminished binding to karyopherin α. Thus,it is concluded that pseudovirion-derived nucleoprotein complexesinteract with karyopherin α in a manner similar to HIV-1 pre-integrationcomplexes.

[0217] The effect of CNI-H0294 on the interaction between karyopherin αand HIV-1 versus pseudovirion nucleoprotein complexes was examined. Itwas found that CNI-H0294 inhibited in a dose-dependent manner binding ofkaryopherin αto HIV-1 pre-integration complexes (FIG. 10C, top panel).Quantitation on a Phosphorimager demonstrated that 0.1 μM and 1 μM ofCNI-H0294 reduced karyopherin α/HIV-1 binding 8- and 25-fold,respectively. These results explain the inhibition of HIV-1 nuclearimport by the compound and correlate well with the dose response curveobtained when HIV-1-infected monocyte cultures were treated withCNI-H0294. Surprisingly, CNI-H0294 did not inhibit binding ofkaryopherin α to pseudovirion-derived nucleoprotein complexes (FIG. 10C,bottom panel) or to purified recombinant MA. These results suggest thatthe mechanism of CNI-0294 inhibition requires a factor(s) present in theHIV-1 pre-integration complex but absent from pseudovirion-derivedcomplexes.

[0218] Structure-activity Relationships Within the CNI-H Group ofCompounds

[0219] To further investigate the mechanism of action of CNI-H0294, thestructure-activity relationships within compounds of the invention wereexamined (Table IX).

[0220] Table IX Structure-Function Analysis of Anti-HIV Activity of CNICompounds

[0221] CNI compounds were added at various concentrations (10 pM to 10nM) to cultures of primary human monocytes together with HIV-1_(ADA) andwere present throughout the entire experiment. A 50% inhibitoryconcentration (IC₅₀) was determined at day 9 after infection. Somecompounds did not achieve 50% inhibition at maximal concentration yetexhibited anti-HIV activity; in these cases the results are presentas >10 μM. Compound Structure IC₅₀ CNI-H0294

50 nM CNI-H1894

>10 μM CNI-H1494

1 μM CNI-H3094

>10 μM

[0222] Absence of the reactive carbonyl groups (compounds CNI-H1494 andCNI-H3094) or the pyrimidine side chain (compound CNI-H1894) resulted ina dramatic decrease of the drug's potency. As the carbonyl groups weredesigned to react with lysine residues within MA NLS, it was notsurprising that their absence decreased the drug's activity. Incontrast, a role for the pyrimidine side chain was unexpected, andsuggested that this side group may be involved in binding CNI-H0294 tothe pre-integration complex.

[0223] CNI-H0294 Binds to RT

[0224] Binding of CNI-H0294 to RT or MA was tested in vitro using[¹⁴C]-labeled CNI-H0294 and recombinant RT and MA proteins (FIG. 11).Specific immunoprecipitation was used to quantify the amount of boundCNI-H0294. Preliminary experiments showed that both anti-RT and anti-MAreagents specifically recognized and immunoprecipitated RT and MA,respectively. As shown in FIG. 11, about 17,000 cpm, or 0.34 nmol ofCNI-H0294 (specific activity 50,000 cpm/nmol) were immunoprecipitablefrom incubations of drug with 0.28 nmol RT, suggesting that CNI-H0294binds to RT in a 1:1 molar ratio. The specificity of this interactionwas further confirmed by immunoprecipitation experiments using coldCNI-H0294 to compete out precipitable counts associated with labeleddrug (FIG. 11). No binding was observed with recombinant MA, and noradioactivity was precipitated by immune sera if the recombinant proteinwas omitted from the reaction mixture. In similar experiments, thebinding of CNI-H0294 to Vpr nor to integrase, two other proteins knownto be present within the HIV-1 pre-integration complex, were detected.These experiments established that CNI-H0294 bound directly to RT, butnot to other proteins of the HIV-1 pre-integration complex. Of interest,CNI-H0294 did not significantly inhibit reverse transcription of HIV-1in infected cells, nor did it block in concentrations up to 50 μM theenzymatic activity of HIV-1 RT in vitro, suggesting that an effect on RTactivity cannot account for the anti-viral action of the compound.

[0225] Binding to RT is Critical for the Anti-HIV Activity of CHI-H0294

[0226] The role of CNI-H0294/RT interaction in the drug's activity wasanalyzed in experiments with compund CNI-H3094. As CNI-H3094 does nothave reactive carbonyl group but contains the active pyrimidine sidechain (see Table IX), it could effectively compete with CHI-H0294 forbinding to the same site on the HIV-1 pre-integration complex, albeit itdid not inhibit nuclear import of HIV-1. FIG. 12A shows that unlabeledCNI-H3094 inhibits binding of [¹⁴C]-labeled CNI-H0294 to RT in adose-dependent manner. Likewise, CNI-H3094 restored binding of HIV-1pre-integration complexes to karyopherin α in the presence CNI-H0294(FIG. 12B). A 5-fold excess of CNI-H3094 (FIG. 12B, lane 4) reducedsignificantly the inhibitory effects of CNI-H0294 on binding of HIV-1pre-integration complexes to karyopherin α, and a 10-fold excess (lane5) completely eliminated the inhibitory effect. In a control experiment,CNI-H3094 did not inhibit binding of HIV-1 pre-integration complexes tokaryopherin α (FIG. 12B, lane 1); this correlates with the compound'slack of anti-HIV activity (Table IX). Finally, CNI-H3094 eliminated theinhibitory effect of CNI-H0294 on HIV-1 replication in monocyte cultures(FIG. 12C). These results confirm the critical role of CNI-H0294/RTinteraction in the drug's mechanism of action and also show a directcorrelation between the drug's binding to RT, inhibition ofHIV-1/karyopherin α interaction, and repression of viral replication.

[0227] CNI-H0294 Inactivates the NLS of MA Without Disrupting MAAssociation with the HIV-1 Genome

[0228] The results presented herein indicate a direct role for RT in theanti-HIV effect of CNI-H0294 and provide a molecular explanation for thehigh specificity of the compound. However, these results do not explainhow CNI-H0294 prevents binding of HIV-1 pre-integration complexes tokaryopherin α, as RT does not bind to directly to karyopherin α. Onepossibility was that binding of CNI-H0294 to RT disrupts thepre-integration complex and causes dissociation of MA from HIV-1 cDNA.

[0229] To test this hypothesis, cytoplasmic lysates of HIV-1-infected H9cells were treated with CNI-H0294 and mixed with immobilized karyopherinα or subjected to immunoprecipitation with antibodies that bind MA.Although CNI-H0294 blocked the interaction of the pre-integrationcomplexes with karyopherin α (FIG. 13A, lane 2), it did not preventimmunoprecipitation of viral DNA with anti-MA serum (FIG. 13A, lane 3);thus MA was still associated with HIV-1 cDNA but lost its ability tobind karyopherin α. As binding of pre-integration complexes tokaryopherin α is controlled mainly by the MA NLS (FIGS. 10A and 10B),these results indicate that CNI-H0294 neutralizes the NLS activity ofMA, either directly (through chemical modification) or indirectly (e.g.by steric hindrance).

[0230] To discriminate between these two possibilities, cytoplasmicextracts of HIV-1-infected cells were treated with [¹⁴C]-CNI-H0294 andthen with sodium borohydride, to reduce the reversible Schiff baseshypothesized to form between the compound and the lysines of MA NLS andconvert the attached drug molecules to irreversible adducts. MA was thenimmunoprecipitated with specific serum in a buffer containing of 0.1%SDS and 1% sodium deoxycholate which disrupts weak protein-drug andprotein-protein interactions in the pre-integration complex withoutdisrupting covalent bonds. Under these conditions, a significant amountof radioactivity was immunoprecipitated by anti-MA serum (FIG. 13B), incontrast to results obtained with recombinant MA (FIG. 11). Theseresults corroborated requirement for RT for the drug's effect andsuggested that the CNI-H0294 had been covalently linked to MA byborohydride treatment. Without borohydride treatment, no radioactivitywas immunoprecipitated with MA. In control experiments, no radioactivitywas precipitated by anti-IN serum, or by anti-MA serum from thecytoplasmic extract of cells infected with pseudovirions (FIG. 13B)which lack RT and thus do not bind CNI-H0294 (FIG. 11).

[0231] CNI-H0294 Inhibits MA NLS⁻, but not Vpr-mediated Binding of HIV-1pre-integration Complexes to Karyopherin α

[0232] Because of the role for MA NLS and Vpr in HIV-1 nuclear import,the effects of CNI-H0294 on the interaction between karyopherin α andpre-integration complexes derived from viruses that carry mutations inVpr (Vpr⁻), MA NLS (MA NLS⁻), or both were investigated (FIG. 11). Theseviruses (except for MA NLS⁻Vpr⁻ double mutant which was slightlyattenuated) entered target cells and reverse transcribed their genomewith similar efficiencies (FIG. 14A). The presence of CNI-H0294diminished binding of karyopherin α to wild-type (wt) (FIG. 14B, lanes1, 2) and Vpr⁻ complexes (lanes 3, 4) by 95% but had no effect onbinding to MA NLS⁻ complexes (lanes 5 and 6) which is only 1-5% of thatobserved with MA NLS+ complexes. The inability of CNI-H0294 to blockbinding of MA NLS⁻ complexes to karyopherin α can be explained by thelack of a consensus NLS in Vpr and that Vpr binds to karyopherin α in anNLS-independent manner.

[0233] Results presented hereinabove reveal the molecular mechanism ofaction of CNI-H0294 that specifically target nuclear import of HIV-1.The mechanism by which CNI-H0294 can inactivate the MA NLS and thusprevent nuclear import of HIV-1 is depicted in FIG. 15. According to theinvention, the compound first binds to HIV-1 pre-integration complexesvia RT and then forms reversible Schiff bases with contiguous lysines inan adjacent MA NLS. This interaction prevents binding of karyopherin αto the MA NLS and significantly inhibits nuclear translocation of theHIV-1 pre-integration complex. The results with pseudovirion-derivednucleoprotein complexes indicate that formation of the functionalcomplex capable of binding karyopherin α and translocating into thenucleus resides entirely within the HIV-1 Gag proteins. Although otherproteins present in the HIV-1 pre-integration complex (e.g. Vpr, IN, RT)may enhance nuclear translocation, they are not necessary for thisprocess. The results indicate that RT and MA NLS are positioned in closeproximity within the HIV-1 pre-integration complex as CNI-H0294 is verysmall yet seems to bind RT and the MA NLS simultaneously. As MA is madefrom the Gag precursor and RT is made from the Gag-Pol precursor, theratio of RT to MA in the virion is expected to be 1:50 because thetranslational frameshift that leads to synthesis of the Gag-Polprecursor (rather than the Gag precursor) occurs about 2% of the time.Interestingly, about 1-2 k of the virion MA protein is phosphorylatedand only these molecules are incorporated into the HIV-1 pre-integrationcomplex. This leads to an important conclusion that there is roughly anequivalent number of RT and MA molecules per HIV-1 pre-integrationcomplex. Given the efficient inactivation of MA NLS by CNI-H0294, mostif not all RT and MA molecules are in close proximity in thepre-integration complex.

[0234] The present invention is not to be limited in scope by thespecific embodiments described which were intended as singleillustrations of individual aspects of the invention, and functionallyequivalent methods and components were within the scope of theinvention. Indeed, various modifications of the invention, in additionto those shown and described herein will become apparent to thoseskilled in the art from the foregoing description and accompanyingdrawings. Such modifications are intended to fall within the scope ofthe appended claims.

We claim:
 1. A method for inactivating a nuclear localization signal ofa protein comprising contacting the protein with a compound that iscapable of stable reversible binding with basic amino acid residues ofthe nuclear localization signal of the protein.
 2. A method forinhibiting importation of a protein into the nucleus of a cellcomprising contacting the protein with a compound that is capable ofstable reversible binding with basic amino acid residues of the nuclearlocalization signal of the protein.
 3. A method for targetedinactivation of a nuclear localization signal of a protein in a complexcomprising contacting the protein with a compound that is capable of:(a) interacting with a molecule in a complex having a specific dockingsite which is positioned proximately to a nuclear localization signal ofa protein in the complex; and (b) forming stable reversible covalentinteractions with basic amino acid residues of the nuclear localizationsignal of the protein.
 4. The method of claim 3 wherein the compoundforms Schiff bases with lysine residues of the nuclear localizationsignal of the protein.
 5. The method of claim 3 wherein the compoundforms stable reversible covalent interactions with arginine residues ofthe nuclear localization signal of the protein.
 6. A method for targetedinactivation of a nuclear localization signal of a protein comprisingcontacting the protein with a compound according to the formula:

wherein A, independently, ═CH₃CH₂CH₃COH, COCH₃COCH₂CH₃, CH₂COCH₃,CH₂COCH₂CH₃, C(CH₃)₂COCH₃, or C(CH₃)₂COCH₂CH₃; P=1 or 2; L is a linkergroup containing an S, O, N or C atom; K=0 or 1; and wherein Jrepresents (i) a saturated or unsaturated, substitued or unsubstituted,straight or branched acyclic hydrocarbon group; (ii) a saturated orsaturated, substitued or unsubstituted, straight or branched acyclicgroup containing hetero atoms such as nitrogen, sulfur or oxygen; (iii)a substituted or unsubstituted, saturated or aromatic, mono- orpoly-cyclic group having 3 to 20 carbon atoms; or (iv) a substituted orunsubstituted, saturated or aromatic, mono- or poly-heterocyclic grouphaving 3 to 20 atoms, at least one of which is a nitrogen, sulfur oroxygen.
 7. A method for targeted inactivation of a nuclear localizationsignal of a protein comprising contacting the protein with a compoundaccording to the formula:

wherein A, independently, ═CH₃, CH₂CH₃, COH, COCH₃, COCH₂CH₃, CH₂COCH₃,CH₂COCH₂CH₃, C(CH₃)₂COCH₃ or C(CH₃)₂COCH₂CH₃; and P=1 or 2; and

wherein X ═NH₂, CH₃ or CH₂CH₃; X′═CH₃ or CH₂CH₃; Y═NH₂, NHCH₃, N(CH₃)₂,1-pyrrolidino or 1-piperidino; and Z=H, CH₃ or CH₂CH₃; or

wherein Y′ and Z′, independently, ═H, NH₂, NHCH₃, N(CH₃)₂, N⁺(CH₃)₃,1-pyrrolidino or 1-piperidino; Q is N or CH; and salts thereof.
 8. Amethod for targeted inactivation of a nuclear localization signal of aprotein comprising contacting the protein with a compound according tothe formula:

wherein A=CH₃, CH₂CH₃, COCH₃, COCH₂CH₃, CH₂COCH₃, CH₂COCH₂CH₃,C(CH₃)₂COCH₃ or C(CH₃)₂COCH₂CH₃; and

wherein X═NH₂, CH₃ or CH₂CH₃; X′═CH₃ or CH₂CH₃; Y═NH₂, NHCH₃, N(CH₃)₂,1-pyrrolidino or 1-piperidino; and Z═H, CH₃ or CH₂CH₃; or

wherein Y′ and Z′, independently, ═H, NH₂, NHCH₃, N(CH₃)₂, N⁺(CH₃)₃ ,1-pyrrolidino or 1-piperidino; Q is N or CH; and salts thereof.
 9. Themethod of claim 3, 4 or 5 wherein the docking site is on the proteinhaving the nuclear localization signal.
 10. The method of claim 3, 4, 5,6, 7 or 8 wherein the protein is derived from a human immunodeficiencyvirus, influenza virus, hepatitis virus, herpes simplex virus,papillomavirus, parvovirus or measles virus.
 11. The method of claim 3wherein the docking site is on the human immunodeficiency virus reversetranscriptase and the nuclear localization signal is in the humanimmunodeficiency virus matrix antigen.
 12. A method for identifyingcompounds that are capable of targeted inactivation of the nuclearlocalization signal of a protein comprising: (a) contacting animmobilized cellular receptor moiety with a protein comprising a nuclearlocalization signal, and a compound having the formula

wherein A, independently, ═CH₃, CH₂CH₃, COH, COCH₃, COCH₂CH₃, CH₂COCH₃,CH₂COCH₂CH₃, C(CH₃)₂COCH₃, or C(CH₃)₂COCH₂CH₃; P=1 or 2; L is a linkergroup containing an S, O, N or C atom; K=0 or 1; and wherein Jrepresents (i) a saturated or unsaturated, substitued or unsubstituted,straight or branched acyclic hydrocarbon group; (ii) a saturated orunsaturated, substitued or unsubstituted, straight or branched acyclicgroup containing hetero atoms such as nitrogen, sulfur or oxygen; (iii)a substituted or unsubstituted, saturated or aromatic, mono- orpoly-cyclic group having 3 to 20 carbon atoms; or (iv) a substituted orunsubstituted, saturated or aromatic, mono- or poly-heterocyclic grouphaving 3 to 20 atoms, at least one of which is a nitrogen, sulfur oroxygen; (b) measuring the binding of the protein to the immobilizedcellular receptor moiety; and (c) comparing the quantity of the proteinbound to the quantity of protein bound in the absence of the compound,where a reduction in the quantity of the bound protein in the presenceof the compound is indicative of targeted inactivation of the nuclearlocalization signal by the compound.
 13. The method of claim 12 whereinthe protein is in a complex.
 14. The method of claim 12 wherein theprotein is derived from a human immunodeficiency virus, influenza virus,hepatitis virus, herpes simplex virus, papillomavirus, parvovirus ormeasles virus.
 15. The method of claim 12 wherein the cellular receptormoiety is karyopherin α.
 16. A method for identifying compounds that arecapable of targeted inactivation of the nuclear localization signal of aviral nucleoprotein complex comprising: (a) contacting an immobilizedkaryopherin α with a viral nucleoprotein complex contained in acytoplasmic extract, said complex comprising viral nucleic acid and saidcytoplasmic extract prepared from cells infected by the virus, and acompound having the formula

wherein A, independently, ═CH₃, CH₂CH₃, COH, COCH₃, COCH₂CH₃, CH₂COCH₃,CH₂COCH₂CH₃, C(CH₃)₂COCH₃, or C(CH₃)₂COCH₂CH₃; P=1 or 2; L is a linkergroup containing an S, O, N or C atom; K=0 or 1; and wherein Jrepresents (i) a saturated or unsaturated, substitued or unsubstituted,straight or branched acyclic hydrocarbon group; (ii) a saturated orunsaturated, substitued or unsubstituted, straight or branched acyclicgroup containing hetero atoms such as nitrogen, sulfur or oxygen; (iii)a substituted or unsubstituted, saturated or aromatic, mono- orpoly-cyclic group having 3 to 20 carbon atoms; or (iv) a substituted orunsubstituted, saturated or aromatic, mono- or poly-heterocyclic grouphaving 3 to 20 atoms, at least one of which is a nitrogen, sulfur oroxygen; (b) measuring the binding of said complex to the immobilizedkaryopherin α by quantitating the amount of viral nucleic acidsassociated with said complex; and (c) comparing the quantity of thenucleic acid bound to the quantity of nucleic acid bound in the absenceof the compound; where a reduction in the quantity of the bound nucleicacid in the presence of the compound is indicative of targetedinactivation of the nuclear localization signal by the compound.
 17. Acompound that is capable of: (a) interacting with a molecule in acomplex having a specific docking site which is positioned proximatelyto a nuclear localization signal of a protein in the complex; and (b)forming stable reversible covalent interactions with basic amino acidresidues of the nuclear localization signal of the protein; and havingthe formula:

 wherein A, independently, ═CH₃, CH₂CH₃, COH, COCH₃, COCH₂CH₃, CH₂COCH₃,CH₂COCH₂CH₃, C(CH₃)₂COCH₃, or C(CH₃)₂COCH₂CH₃; P=1 or 2; L is a linkergroup containing an S, O, N or C atom; K=0 or 1; and wherein Jrepresents (i) a saturated or unsaturated, substitued or unsubstituted,straight or branched acyclic hydrocarbon group; (ii) a saturated orunsaturated, substitued or unsubstituted, straight or branched acyclicgroup containing hetero atoms such as nitrogen, sulfur or oxygen; (iii)a substituted or unsubstituted, saturated or aromatic, mono- orpoly-cyclic group having 3 to 20 carbon atoms; or (iv) a substituted orunsubstituted, saturated or aromatic, mono- or poly-heterocyclic grouphaving 3 to 20 atoms, at least one of which is a nitrogen, sulfur oroxygen.
 18. The compound of claim 17 wherein the protein is derived froma virus.
 19. The compound of claim 17 wherein the protein is derivedfrom a human immunodeficiency virus, influenza virus, hepatitis virus,herpes simplex virus, papillomavirus, parvovirus or measles virus.
 20. Amethod of preventing productive infection by a virus of a proliferatingpopulation of cells, which comprises preventing importation of a complexcontaining viral nucleic acid or viral protein into the nucleus of acell in the population.
 21. The method of claim 20 which furthercomprises the administration of an effective amount of a pharmaceuticalcomposition containing a compound according to the formula:

wherein A, independently, ═CH₃, CH₂CH₃, COH, COCH₃, COCH₂CH₃, CH₂COCH₃,CH₂COCH₂CH₃, C(CH₃)₂COCH₃, or C(CH₃)₂COCH₂CH₃; P=1 or 2; L is a linkdergroup containing an S, O, N or C atom; K=0 or 1; and wherein Jrepresents (i) a saturated or unsaturated, substitued or unsubstituted,straight or branched acyclic hydrocarbon group; (ii) a saturated orunsaturated, substitued or unsubstituted, straight or branched acyclicgroup containing hetero atoms such as nitrogen, sulfur or oxygen; (iii)a substituted or unsubstituted, saturated or aromatic, mono- orpoly-cyclic group having 3 to 20 carbon atoms; or (iv) a substituted orunsubstituted, saturated or aromatic, mono- or poly-heterocyclic grouphaving 3 to 20 atoms, at least one of which is a nitrogen, sulfur oroxygen.
 22. The method of claim 20 which comprises the administration ofan effective amount of a pharmaceutical composition containing CompoundNo. 2 as an active ingredient.
 23. The method of claim 1 wherein thecompound is capable of forming tandem Schiff bases with lysine residuesof the nuclear localization signal of the protein.
 24. The method ofclaim 1 wherein the compound is capable of forming stable reversibleadducts with arginine residues of the nuclear localization signal of theprotein.
 25. The method of claim 3, 4, 5, 6, 7 or 8 wherein the proteinis a transcription factor.